Selective Caspase Inhibitors and Uses Thereof

ABSTRACT

The present invention relates to compounds of Formula I, IA, II, HA, III, or IHA and their pharmaceutical uses. Particular aspects of the invention relate to the use of those compounds for the selective inhibition of one or more caspases. Also described are methods where the compounds of Formula I, IA, II, IIA, III, or IIIA are used in the prevention and/or treatment of various diseases and conditions in subjects, including caspase-mediated diseases such as sepsis, myocardial infarction, ischemic stroke, spinal cord injury (SCI), traumatic brain injury (TBI) and neurodegenerative disease (e.g. multiple sclerosis (MS) and Alzheimer&#39;s, Parkinson&#39;s, and Huntington&#39;s diseases).

FIELD OF INVENTION

The present invention relates to chemical compounds and theirpharmaceutical uses. More particularly, the invention relates toselective inhibitors of caspases and their uses for the preventionand/or treatment of various diseases and conditions in subjects.

BACKGROUND OF INVENTION

Caspases comprise a family of cysteine protease enzymes with awell-known role as key mediators in apoptosis signaling pathways andcell disassembly. Interleukin converting enzyme (ICE), also known asCaspase-1, was the first identified caspase. In humans, 11 other knowncaspases have been further identified. Caspases have been classified intwo general groups according to their effects: proapoptotic (caspase-2,3, 6, 7, 8, 9, 10) and proinflammatory (caspase-1, 4, 5, 11, 12)caspases. The proapoptotic caspases have been divided in initiators(caspase-2, 8, 9, 10) also known as group II, and executioners(caspase-3,6,7) of the apoptotic process or group III. The Interleukinconverting enzyme (ICE) also known as Caspase-1 has a proinflammatoryrole only.

There is growing evidence demonstrating the role of caspases in verydiverse pathologies. For instance it is known that proapoptotic caspasesare involved in the pathogenesis of many cardiovascular disorders. Someproapoptotic caspases such as caspase-8 also possess non-apoptoticfunction that may contribute to tumor progression. Caspase-1 plays animportant role in response to pathogenic infection as well as ininflammatory and autoimmune disorders. In addition, caspase-1 activityis increased in retinas of diabetic patients and it constitutes acritical regulator of cardiomyocyte programmed cell death in themammalian heart. Caspases also plays a role in neurodegenerativediseases and trauma. For instance, it has been shown that the caspase-3cascade is highly activated due to the traumatic spinal cord injury.Finally, the activation of caspase-1 and caspase-3 in AmyotrophicLateral Sclerosis (ALS) patients and the activation of caspase-7, -8,and -9 in a mouse model at end stage of ALS have been reported.Increased levels of apoptosis and caspase activity (especiallycaspase-3) are reported to be frequently observed at sites of cellulardamage in both acute (e.g. Sepsis, myocardial infarction(MI), IschemicStroke, Spinal cord injury (SCI), traumatic Brain Injury (TBI)) andneurodegenerative disease (e.g. Alzheimer's, Parkinson's andHuntington's diseases, and multiple sclerosis (MS)).

Since caspases are involved in a number of diseases, several compoundsand methods have been developed to inactivate them. For example, thebroad irreversible caspase inhibitorbenzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) wasprotective and efficiently blocked death receptor-mediated liver injuryin animal models (Rodriguez et al. (1996), J Exp Med. 1996 Nov. 1;184(5):2067-72). Myocardial infarction and the resulting death ofmyocytes was ameliorated by z-VAD-fmk and related peptide inhibitors inanimal models (Yaoita et al., 91998) Circulation 97: 276-281). Therehave been also a lot of efforts for identifying inhibitors of peptidase.For instance, Hanzlik and Thompson (J. Med. Chem. (1984), 27(6),711-712) describe vinylogous amino acid esters for inactivating thiolproteases. Thompson et al. (J. Med. Chem. (1986), 29(1), 104-111)describe carboxyl-modified amino acids and peptides as proteaseinhibitors. Liu and Hanzlik have prepared a series of peptidyl Michaelacceptors with different electron withdrawing groups with differentrecognition and binding groups as inactivators against papain, a memberof the cysteine proteinase family. Similarly, U.S. Pat. Nos. 5,976,858and 6,287,840 to Palmer wt et al. describes irreversible cysteineprotease inhibitors containing vinyl groups conjugated to electronwithdrawing groups. However, these and other compounds are not effectiveagainst caspases, because caspases are among the most specificendopeptidases.

Given their role in several diseases and conditions, there is a need forcompounds capable of selectively targeting either a specific caspase ora group of caspases. There is also a need for effective pharmaceuticalcompositions and method of treatment for caspase-related diseases.

The present invention addresses these needs for novel therapies, newtreatment methods, compounds, and pharmaceutical compositions.

Additional features of the invention will be apparent from review of thedisclosure, figures and description of the invention below.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to compounds according to any of FormulaI, IA, II, IIA, III, or IIIA as defined herein, compositions thereof andmethods for the prevention and/or treatment of caspase-related diseasesin subjects. Particular aspects of the invention relates to use ofcompounds according to any of Formula I, IA, II, IIA, III, or IIIA asdefined herein.

One aspect of the invention concerns a method for preventing and/ortreating a caspase-related disease in a subject in need thereof,comprising administering to said subject an effective amount of acompound represented by any of Formula I, IA, II, IIA, III, or IIIA asdefined herein.

One aspect of the invention concerns the use of a compound a compoundrepresented by any of Formula I, IA, II, IIA, III, or IIIA as definedherein for preventing and/or treating of caspase-related diseases in asubject in need thereof.

Another related aspect of the invention concerns the use of a compoundrepresented by any of Formula I, IA, II, IIA, III, or IIIA as definedherein for the manufacture of a medication for preventing and/ortreating of caspase-related diseases in a subject in need thereof.

One aspect of the invention concerns a method of treating excessiveapoptosis affected by caspase activity in a cell or a tissue, the methodcomprising: contacting the cell or tissue with an effective amount ofone or more compounds represented by any of Formula I, IA, II, IIA, III,or IIIA as defined herein, so as to treat the excessive apoptosis.

One particular aspect of the invention concerns the use of a compoundrepresented by any of Formula I, IA, II, IIA, III, or IIIA as definedherein for use in apoptosis mediated diseases.

Caspase-related disease as defined herein are selected from the groupconsisting of apoptosis mediated diseases, IL-1 mediated diseases,inflammatory diseases, autoimmune diseases, autoinflammatory diseases,proliferative diseases, infectious diseases, degenerative diseases,retinal disorders, inflammatory peritonitis, osteoarthritis,pancreatitis, asthma, respiratory distress syndrome, rheumatoidarthritis, systemic lupus erythematous, scleroderma, Grave's disease,autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmuneneutropenia, thrombocytopenia, hepatitis, inflammatory bowel disease,crohn's disease, psoriasis, dermatitis, Graft vs host disease, organtransplant rejection, osteoporosis, leukemias and related disorders,multiple myeloma-related diseases, metastatic melanomas, Kaposi'ssarcoma, sepsis, septic shock, Alzheimer's disease, Parkinson's disease,Huntington's disease, cerebral ischemia, epilepsy, myocardial ischemia,acute and chronic heart disease, myocardial infarction, congestive heartfailure, atherosclerosis, spinal muscular atrophy, amyotrophic lateralsclerosis, multiple sclerosis, HIV-related encephalitis, aging,neurological damage due to stroke, ulcerative colitis, traumatic braininjury, spinal cord injury, hepatitis-B, hepatitis-C, hepatitis-G,liver-related diseases, renal disease, and HIV infection.

Specific examples of compounds according to the invention arerepresented in Table 1.

The invention also provides methods and strategies of targetingcaspases. In one embodiment the approach consists of designing a suicidesubstrate leading to a permanent inhibition of the caspase. Preferably,the approach consists of designing a substrate that is recognizableenough for caspases, especially one or more specific caspase(s), to fitinto it, to be potentially cleaved at a specific position in a way thatmakes the caspase enzyme irreversibly linked to the substrate therebyleading to a permanent inhibition of the caspase. In some embodiments,the suicide substrates of this invention are vinyl electron withdrawinggroup (EWG).

Further aspects of the invention will be apparent to a person skilled inthe art from the following description, and claims and generalizationstherein.

DETAILED DESCRIPTION OF THE INVENTION A) General Overview of theInvention

The present inventors have discovered compounds that have beneficialpharmaceutical properties and that these compounds may be effective foruse in caspase-mediated diseases such as sepsis, myocardial infarction,ischemic stroke, spinal cord injury (SCI), traumatic brain injury (TBI)and neurodegenerative disease (e.g. multiple sclerosis (MS) andAlzheimer's, Parkinson's, and Huntington's diseases).

B) Compounds of the Invention

Broadly speaking, the invention concerns a compound represented byFormula I

wherein A, PX, P5, P4, P3, P2, R¹, R², a and b are as definedhereinabove and hereinbelow; or a prodrug, or a pharmaceuticallyacceptable salt to allow the drug to penetrate the cell membrane or thecompound is labeled with a detectable label or an affinity tag thereof.

The line “-” when located between P2, P3, P4, P5 and PX represents apeptide bond or a peptidomimetic bond; The PX, P5, P4, P3, P2 amino acidresidues are normally linked via a peptide bond, that is, a peptidiccarbamoyl group, i.e. —CONH—. However, peptidomimetic bonds are alsocontemplated, such as CH₂—NH, CO—CH₂, azapeptide and retro-inversobonds.

The R¹ and R² that are bonded to the vinyl group can be either in thecis configuration or the trans configuration, as represented by the wavylines. In one example, R¹ is configured to be trans such that theelectron withdrawing capability of the R¹ group is stabilized.

Further included within the scope of the invention are compounds ofFormula IA:

wherein A, AA_(X), AA₅, AA₄, AA₃, AA₂, R¹, R², a and b are as definedhereinabove and hereinbelow;or a prodrug, or a pharmaceutically acceptable salt to allow the drug topenetrate the cell membrane, or the compound is labeled with adetectable label or an affinity tag thereof.

Thus, when a and b are both 0, the present invention includes compoundsof Formula II:

wherein A, AA₄, AA₃, AA₂, R¹, and R² are as defined hereinabove andhereinbelow. Furthermore, when a is 0 and b is 1, the present inventionincludes compounds of Formula III

wherein A, AA₅, AA₄, AA₃, AA₂, R¹, and R² are as defined hereinabove andhereinbelow. One subset of compounds of Formula II includes compounds ofFormula IIA:

wherein A, AA₄, AA₃, AA₂, R¹, and R² are as defined hereinabove andhereinbelow.

One subset of compounds of Formula III includes compounds of FormulaIIIA:

wherein A, AA₅, AA₄, AA₃, AA₂, R¹, and R² are as defined hereinabove andhereinbelow.a and b:In one subset of compounds of the invention, a is 0 or 1; and b is 0 or1 provided that when b is 0, a is 0.In one example, a and b are both 0.In another example, a is 0 and b is 1.

A:

In one subset, A is

-   -   1) H,    -   2) C₁-C₆ alkyl,    -   3) aryl,    -   4) heteroaryl,    -   5) heterocyclyl,    -   6) R³—OC(O)—;    -   7) R³—C(O)O—, or    -   8) R³—S(O)₂—;        wherein R³ is    -   1) C₁-C₆ alkyl,    -   2) aryl,    -   3) heteroaryl, or    -   4) heterocyclyl;        In one example, A is H.        In one example, A is R³—OC(O)—.        In one example, A is PhCH₂OC(O)—.

R¹:

In one subset, R¹ is an electron withdrawing group (EWG) selected from

-   -   1) aryl,    -   2) heteroaryl,    -   3) heterocyclyl,    -   4) C₂-C₆ alkene-R²⁰,    -   5) SO₂R⁵,    -   6) SO₃R⁵,    -   7) SOR⁵,    -   8) SONHR⁵,    -   9) SO₂NHR⁵,    -   10) CN,    -   11) CO₂R⁵,    -   12) COR⁵,    -   13) PO₃R⁵,    -   14) PO(OR⁵)₂, or    -   15) PO(OR⁵),        wherein the aryl, the heteroaryl, or the heterocyclyl are        optionally substituted with one or more R³⁰.

R²:

In one subset, R² is

-   -   1) R¹; or    -   2) H,    -   3) halogen,    -   4) haloalkyl,    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkene,    -   7) C₃-C₇ cycloalkyl,    -   8) OR⁹;    -   9) OCOR⁶,    -   10) OCO₂R⁶,    -   11) NR⁷R⁸,    -   12) NHSO₂R⁶,    -   13) NHCOR⁶,    -   14) aryl,    -   15) heteroaryl, or    -   16) heterocyclyl;        wherein R⁶, R⁷, R⁶ and R⁹ are as defined hereinabove and        hereinbelow.        In one example, R² is H.        In another example, R² is halogen.        In yet another example, R² is Cl.

R⁴:

In one subset, R⁴ is

-   -   1) H, or    -   2) methyl, ethyl, propyl, or tert-butyl.        In one example, R⁴ is H.

R⁵:

In one subset, R⁵ is

-   -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkene,    -   4) C₃-C₇ cycloalkyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl, or    -   8) any optionally protected (D) or (L) amino acid residue.

R⁶:

In one subset, R⁶ is

-   -   1) any (D) or (L) amino acid residue,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) aryl,    -   5) heteroaryl, or    -   6) heterocyclyl,        in which the alkyl or the cycloalkyl are optionally substituted        with one or more R¹⁶ substituents; and in which the aryl,        heteroaryl or heterocyclyl are optionally substituted with one        or more R²⁰ substituents.

R⁷ and R⁸:

In one subset, R⁷ and R⁸ are independently selected from:

-   -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) heteroaryl, or    -   7) heterocyclyl,        wherein the alkyl and the cycloalkyl are optionally substituted        with one or more R¹⁰ substituents, and the aryl, the heteroaryl        and the heterocyclyl are optionally substituted with one or more        R²⁰ substituents.

R⁹:

In one subset, R⁹ is

-   -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) aryl,    -   5) heteroaryl, or    -   6) heterocyclyl,        in which the alkyl or the cycloalkyl are optionally substituted        with one or more R¹⁹ substituents; and in which the aryl,        heteroaryl or heterocyclyl are optionally substituted with one        or more R²⁰ substituents.

R¹⁰:

In one subset, R¹⁰ is independently selected from:

-   -   1) halogen,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl,    -   8) OR⁹,    -   9) S(O)_(m)R⁹,    -   10) NR⁷R⁸,    -   11) COR⁵,    -   12) C(O)OR⁹,    -   13) OC(O)R⁹,    -   14) SC(O)R⁹,    -   15) CONR⁷R⁸, or    -   16) S(O)₂NR⁷R⁸;        wherein R⁷, R⁸, R⁹ are as defined hereinabove and hereinbelow;        m is an integer of 0, 1, or 2.

R²⁰:

In one subset, R²⁰ is independently selected from:

-   -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) C₁-C₆ alkyl,    -   5) haloalkyl,    -   6) C₃-C₇ cycloalkyl,    -   7) OR⁷,    -   8) NR⁷R⁸,    -   9) SR⁷,    -   10) aryl,    -   11) heteroaryl,    -   12) heterocyclyl,    -   13) SO₂R⁵,    -   14) SO₃R⁵,    -   15) SOR⁵,    -   16) SONHR⁵,    -   17) SO₂NHR⁵,    -   18) PO₃R⁵,    -   19) PO(OR⁵)₂,    -   20) PO(OR⁵),    -   21) COR⁷,    -   22) CO₂R⁷,    -   23) S(O)_(m)R⁷,    -   24) CONR⁷R⁸, or    -   25) S(O)₂NR⁷R⁸,        wherein the alkyl and the cycloalkyl are optionally substituted        with one or more R⁶ substituents; and wherein the aryl, the        heteroaryl, or the heterocyclyl are optionally substituted with        one or more R³⁰.        wherein R⁷, R⁸ and m are as defined hereinabove and hereinbelow.

R³⁰:

In one subset, R³⁰ is

-   -   1) NO₂,    -   2) C₂-C₆ alkene-R²⁰,    -   3) SO₂R⁵,    -   4) SOR⁵,    -   5) SONHR⁵,    -   6) SO₂NHR⁵,    -   7) CN,    -   8) CO₂R⁵,    -   9) COR⁵,    -   10) PO₃R⁵,    -   11) PO(OR⁵)₂, or    -   12) PO(OR⁵);        wherein R⁵ and R²⁰ are as defined hereinabove and hereinbelow.

Caspase 3 Inhibitors

The present invention includes compounds of Formula IIA:

whereinAA₂ is the amino acid side chain of Val, Leu, Pro, Met, Ala, Thr, His.AA₃ is the amino acid side chain of Trp, Tyr, Ala, Asp, Glu, Gln, Phe,Ser, Thr, Val, Tyr, Gly, Leu; or AA₃ is phenylglycine, indanylglycine,or Ala-(2′-quinolyl);AA₄ is the amino acid side chain of Asp;and wherein A, R¹, R² and R⁴ are as defined hereinabove and hereinbelow.

Caspase 8/Caspase 9 Inhibitors

The present invention includes compounds of Formula IIA:

whereinAA₂ is the amino acid side chain of Thr, His, Val, Trp, Ile, or AlaAA₃ is the amino acid side chain of Glu or AA₃ is Ala-(2′-quinolyl);AA₄ is the amino acid side chain of Ile, Leu, Glu, Asp, Ala, Pro or Val;and wherein A, R¹, R² and R⁴ are as defined hereinabove and hereinbelow.

Caspase 2 Inhibitors

The present invention includes compounds of Formula IIIA

whereinAA₂ is the amino acid side chain of Ala, Ser, Lys or Val;AA₃ is the amino acid side chain of Val, Glu, Thr, or Gln;AA₄ is the amino acid side chain of Asp, or Leu;AA₅ is the amino acid side chain of Val or Leu;and wherein A, R¹, R² and R⁴ are as defined hereinabove and hereinbelow.

Caspase 1 Inhibitors

The present invention includes compounds of Formula IIA (caspase 1inhibitors)

whereinAA₂ is the amino acid side chain of Val, Ala, Thr, or His;AA₃ is the amino acid side chain of Glu, Gln, Asp, Ala, Gly, Thr, Val,Trp; or AA₃ is phenylglycine or indanylgiycine;AA₄ is the amino acid side chain of Tyr, Trp, Phe, or Asp;and wherein A, R¹, R² and R⁴ are as defined hereinabove and hereinbelow.

Compounds and intermediate compounds synthesized according to thepresent invention include those in Table 1:

TABLE 1 CPD N° CPD NAME STRUCTURE 1 Fmoc-Ala(2′-quinolyl)-Val-OAllyl

2 Ala(2′quinolyl)-Val-OAllyl

3 Cbz-Asp(O-tBu)-Ala(2′-quinolyl)- Val-OAllyl

4 Cbz-Asp(O-tBu)-Ala(2′-quinolyl)- ValOH

5 Ts-Ala(2′-quinolyl)-Val-OH

6 Ts-Ala(2′-quinolyl)-OH

7 Ts-Ala(2′-quinolyl)-Val-OAllyl

8 Z-Asp(OtBu)-Tyr(OtBu)-Val- Asp(OtBu)methyl vinyl sulfone

9 Fmoc-Indanylglycine-Val-OAllyl

10 Indanylglycine-Val-OAllyl

11 Cbz-Asp(O-tBu)-Indanylglycine- Val-OAllyl

12 Cbz-Asp(O-tBu)Indanylglycine- Val-OH

13 Fmoc-Phg-Val-OAllyl

14 Phg-Val-OAllyl

15 Z-Asp(β-tert-Butyl)-Phg-Val-Oallyl.

16 Z-Asp(β-tert-Butyl)-Phg-Val-OH.

17 Fmoc-Glu(O-tBu)-Val-OAllyl

18 Glu(O-tBu)-Val-OAllyl

19 Cbz-Asp(O-tBu)-Glu(O-tBu)- Val-OAllyl

20 Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-OH

21 Diethyl chloro(methylsulfone) methylphosphonate

22 Boc-Asp (β-tert-butyl) αchlorovinyl methylsulfone

23 Asp(β-tert-butyl)αchlorovinyl methylsulfone tosyl salt

24 Diethyl chloro(phenylsulfone)methyl- phosphonate

25 Asp(β-tert-butyl)αchlorovinyl phenylsulfone

26 Asp(β-tert-butyl)αchlorovinyl phenylsulfone tosyl salt

27 Boc-Aspartimol(β-Methyl)

28 Boc-Asp(β-Methyl)-H

29 Boc-Asp(β-Methyl)methyl vinyl sulfone

30 Boc-Asp(β-Methyl)methyl vinyl sulfone tosyl salt

31 Diethyl (methylsulfone)methylphosphonate

32 Boc-Asp(β-tert-butyl)methyl vinyl sulfone

33 Asp(β-tert-butyl)methyl vinyl sulfone tosyl salt

34 Diethyl phenylsulfonylmethylphosphonate

35 Boc-Asp(β-tert-Butyl)-H

36 Boc-Asp-vinyl phenyl sulfone

37 AspVinyl phenyl sulfone tosyl salt

38 Diethyl (phenoxysulfonyl)methylphosphonate

39 Asp(β-tert-butyl)phenoxy vinyl sulfone

40 Asp(β-tert-butyl)phenoxy vinyl sulfone tosyl salt

41 Diethyl (isopropylsulfone)methylphosphonate

42 BocAsp(β-tert-butyl)isopropyl vinyl sulfone

43 Asp(β-tert-butyl)isopropyl vinyl sulfone tosyl salt

44 Diethyl (morpholinesulfone)methyl phosphonate

45 Boc-Asp(β-tert-butyl)morpholine vinyl sulfone

46 Asp(β-tert-butyl)morpholine vinyl sulfone tosyl salt

47 Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)αchlorovinyl methylsulfone

48 Z-Asp-Ala(2′-quinolyl)-Val-Asp- αchlorovinyl methylsulfone

49 Ts-Ala(2′quinolyl)-Val-Asp(β-tert- Butyl)-αchlorovinyl methylsulfone

50 Ts-Ala(2′quinolyl)-Val-Asp- αchlorovinyl methylsulfone

51 Z-Asp(β-methyl)-Indanylglycine-Val- Asp(β-methyl)methyl vinyl sulfone

52 Z-Asp(β-tert-Butyl)-Phg-Val-Asp(β- tert-Butyl)methyl vinyl sulfone

53 Z-Asp-Phg-Val- Aspmethyl vinyl sulfone

54 Z-Asp(β-tert-Butyl)-Al(2′-quinolyl)- Val-Asp(β-tert-Butyl)methylvinyl sulfone

55 Z-Asp-Ala(2′-quinolyl)-Val- Aspmethyl vinyl sulfone

56 Z-Asp(β-tert-Butyl)-Indanylglycine- Val-Asp(β-tert-Butyl)methyl vinylsulfone

57 Z-Asp-Indanylglycine-Val- Aspmethyl vinyl sulfone

58 Z-Asp(β-tert-Butyl)- Glu(β-tert-Butyl)- Val-Asp(β-tert-Butyl)methylvinyl sulfone

59 Z-Asp-Glu-Val-Aspmethyl vinyl sulfone

60 Z-Val-Asp(β-tert-Butyl)methyl vinyl sulfone

61 Z-Val-Aspmethyl vinyl sulfone

62 Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)- Val-Asp(β-tert-Butyl)phenylvinyl sulfone

63 Z-Asp-Ala(2′-quinolyl)-Val-Aspphenyl vinyl sulfone

64 Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)- Val-Asp(β-tert-Butyl)phenoxyvinyl sulfone

65 Z-Asp-Ala(2′-quinolyl)-Val- Aspphenoxy vinyl sulfone

66 Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)- Val-Asp(β-tert-Butyl)morpholinevinyl sulfone

67 Z-Asp-Ala(2′-quinolyl)-Val- Aspmorpholinevinyl sulfone

68 Z-Asp-Indanylglycine-Val- Aspisopropyl vinyl sulfone

69 Z-Asp-Phg-Val-Asp-phenyl vinylsulfone

70 Z-Asp-(D,L Ala(2′-quinolyl))-Val- Aspphenyl vinylsulfone

71 Z-Asp-(D,L Ala(2′-quinolyl))-Val- Aspmethyl vinylsulfone

72 Fmoc-Tyr(O-tBu)-Val-Oallyl

73 Tyr(O-tBu)-Val-Oallyl

74 Z-Asp(O-tBu)-Tyr(O-tBu)-Val-Oallyl

75 Z-Asp-Tyr(O-tBu)-Val-OH

76 Z-Asp-Tyr-Val-Aspmethyl vinyl sulfone

77 Fmoc-Glu(O-tBu)-Val-Oallyl

78 Glu(O-tBu)-Val-Oallyl

79 Z-Tyr(O-tBu)-Glu(O-tBu)-Val-Oallyl

80 Z-Tyr(O-tBu)-Glu(O-tBu)-Val-OH

81 Z-Tyr(O-tBu)-Glu(O-tBu)-Val- Asp(OtBu)methyl vinyl sulfone

82 Z-Tyr-Glu-Val-Aspmethyl vinyl sulfone

83 Z-Asp(O-tBu)-Ala-(2′pyridine)-Val- OH

84 Z-Asp(O-tBu)-Ala-(2′pyridine)-Val- Asp(O-tBu)phenyl vinyl sulfone

85 Z-Asp-Ala(2′-pyridnyl)-Val- Aspphenyl vinylsulfone

86 Z-Asp(O-tBu)-Trp-Val-OH

87 Z-Asp(O-tBu)-Trp-Val-Asp(OtBu) methyl vinyl sulfone

88 Z-Asp-Trp-Val-Aspmethyl vinyl sulfone

89 Boc-Asp(β-methyll)αchlorovinyl methylsulfone

90 Asp(β-methyll)αchlorovinyl methylsulfone tosyl salt

91 Boc-Asp(β-methyll)αmethoxyvinyl methysulfone

92 Asp(β-methyll)αmethoxyvinyl methylsulfone tosyl salt

93 Aspmethyl vinyl sulfone tosyl salt

94 Z-Tyr(OtBu)-Val-Ala-OH

95 Z-Tyr(OtBu)-Val-Ala-Asp(OtBu) phenyl vinyl sulfone

96 Z-Tyr-Val-Ala-Asp phenyl vinyl sulfone

DEFINITIONS

Unless otherwise specified, the following definitions apply:

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that the listof elements following the word “comprising” are required or mandatorybut that other elements are optional and may or may not be present.

As used herein, the term “consisting of” is intended to mean includingand limited to whatever follows the phrase “consisting of”. Thus thephrase “consisting of” indicates that the listed elements are requiredor mandatory and that no other elements may be present.

As used herein, the term “alkyl” is intended to include both branchedand straight chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, for example, C₁-C₆ as in C₁-C₆-alkylis defined as including groups having 1,2,3,4,5 or 6 carbons in a linearor branched arrangement. Examples of C₁-C₆-alkyl and C₁-C₄ alkyl asdefined above include, but are not limited to, methyl, ethyl, n-propyl,i-propyl, n-butyl, t-butyl, i-butyl, pentyl and hexyl.

As used herein, the term, “alkenyl” is intended to mean unsaturatedstraight or branched chain hydrocarbon groups having the specifiednumber of carbon atoms therein, and in which at least two of the carbonatoms are bonded to each other by a double bond, and having either E orZ regiochemistry and combinations thereof. For example, C₂-C₆ as inC₂-C₆ alkenyl is defined as including groups having 1, 2, 3, 4, 5, or 6carbons in a linear or branched arrangement, at least two of the carbonatoms being bonded together by a double bond. Examples of C₂-C₆ alkenylinclude ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl and the like.

As used herein, the term “cycloalkyl” is intended to mean a monocyclicsaturated aliphatic hydrocarbon group having the specified number ofcarbon atoms therein, for example, C₃-C₇ as in C₃-C₇ cycloalkyl isdefined as including groups having 3,4,5,6, or 7 carbons in a monocyclicarrangement. Examples of C₃-C₇ cycloalkyl as defined above include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

As used herein, the term “halo” or “halogen” is intended to meanfluorine, chlorine, bromine and iodine.

As used herein, the term “haloalkyl” is intended to mean an alkyl asdefined above, in which each hydrogen atom may be successively replacedby a halogen atom. Examples of haloalkyls include, but are not limitedto, CH₂F, CHF₂ and CF₃.

As used herein, the term “aryl”, either alone or in combination withanother radical, means a carbocyclic aromatic monocyclic groupcontaining 6 carbon atoms which may be further fused to a second 5- or6-membered carbocyclic group which may be aromatic, saturated orunsaturated. Aryl includes, but is not limited to, phenyl, indanyl,1-naphthyl, 2-naphthyl and tetrahydronaphthyl. The aryls may beconnected to another group either at a suitable position on thecycloalkyl ring or the aromatic ring.

As used herein, the term “heteroaryl” is intended to mean a monocyclicor bicyclic ring system of up to ten atoms, wherein at least one ring isaromatic, and contains from 1 to 4 hetero atoms selected from the groupconsisting of O, N, and S. The heteroaryl substituent may be attachedeither via a ring carbon atom or one of the heteroatoms. Examples ofheteroaryl groups include, but are not limited to thienyl,benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl,isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl,thiazolo[4,5-b]-pyridine, and fluoroscein derivatives.

As used herein, the term “heterocycle”, “heterocyclic” or “heterocyclyl”is intended to mean a 5, 6, or 7 membered non-aromatic ring systemcontaining from 1 to 4 heteroatoms selected from the group consisting ofO, N and S. Examples of heterocycles include, but are not limited topyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl,imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, andpyrazolinyl.

As used herein, the term “electron withdrawing group (EWG)” is intendedto mean a functional group that allows nucleophilic attack by thethiol-group of a caspase at the alkene bond of the inhibitor as a resultof the electron withdrawing properties of the EWG. The EWG is conjugatedwith the alkene bond, such that the electron withdrawing properties ofthe EWG allow nucleophilic attack by a caspase at the alkene bond, i.e.the alkene bond and the EWG are electronically conjugated. Thus, thecovalent bond between the alkene bond and the EWG is a direct one,without intervening moieties that would prevent the electron withdrawingproperties of the EWG from being exerted on the alkene bond.

As used herein, the term “detectable label” is intended to mean a groupthat may be linked to a compound of the present invention to produce aprobe or to a caspase, such that when the probe is associated with thecaspase, the label allows either direct or indirect recognition of theprobe so that it may be detected, measured and quantified.

As used herein, the term “affinity tag” is intended to mean a ligand orgroup, which is linked to either a compound of the present invention orto a caspase to allow another compound to be extracted from a solutionto which the ligand or group is attached.

As used herein, the term “probe” is intended to mean a compound ofFormula I, IA, II, IIA, III, or IIIA, which is labeled with either adetectable label or an affinity tag, and which is capable of binding,either covalently or non-covalently, to a caspase. When, for example,the probe is non-covalently bound, it may be displaced by a testcompound. When, for example, the probe is bound covalently, it may beused to form cross-linked adducts, which may be quantified and inhibitedby a test compound.

As used herein, the term “optionally substituted with one or moresubstituents” or its equivalent term “optionally substituted with atleast one substituent” is intended to mean that the subsequentlydescribed event of circumstances may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. The definition is intended to meanfrom zero to five substituents.

If the substituents themselves are incompatible with the syntheticmethods of the present invention, the substituent may be protected witha suitable protecting group (PG) that is stable to the reactionconditions used in these methods. The protecting group may be removed ata suitable point in the reaction sequence of the method to provide adesired intermediate or target compound. Suitable protecting groups andthe methods for protecting and de-protecting different substituentsusing such suitable protecting groups are well known to those skilled inthe art; examples of which may be found in T. Greene and P. Wuts,Protecting Groups in Chemical Synthesis (3^(rd) ed.), John Wiley & Sons,NY (1999), which is incorporated herein by reference in its entirety.Examples of protecting groups used throughout include, but are notlimited to Fmoc, Bn, Boc, CBz and COCF₃. In some instances, asubstituent may be specifically selected to be reactive under thereaction conditions used in the methods of this invention. Under thesecircumstances, the reaction conditions convert the selected substituentinto another substituent that is either useful in an intermediatecompound in the methods of this invention or is a desired substituent ina target compound.

Three and single letter abbreviations for α-amino acids used throughoutare as follows:

Amino acid Abbreviation Abbreviation α-Amino butyric acid Abu — AlanineAla A Arginine Arg R Aspartic acid Asp D Asparagine Asn N Cysteine Cys CGlutamic acid Glu E Glutamine Gln Q Glycine Gly G Isoleucine Ile IHistidine His H Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val VA series of non natural amino acids which may be used in place ofnatural amino acids is but not limited to 3-amino 2-hydroxy pyridine;(2furyl)alanine; 1-amino 1cyclohexane carboxylic acid;(2-thienyl)alanine; 2-Aminobenzoic acid (2-Abz); 2PyridylAlanine; 1Amino1Cyclopentanecarboxilic acid; 2-Aminobutyric acid (2Abu);3Amino3phenylpropionic acid; Aminocyclopentane carboxylic acid (ACPC);4-Aminomethylbenzoic acid (Amb); Aminoisobutiric acid (Aib);p-Benzoyl-1-phenylalanine (Bpa); AllylGlycine; 4-Aminomethyl cyclohexanecarboxylic acid (Amc); Cyclohexyl-alanine (Cha); deltaValine;deltaLeucine; Cyanobbutylalanine (Cba); Indanylglycine (Igl);3-(2-naphthyl)alanine (1-Nal); Biphenylalanine (Bip); Hydroxyproline(Hyp); Isonipecotic acid (Inp); Norvaline (Nva); 4-Iodophenylalanine(Phe(pl)); 4-nitroPhenylalanine; 4MethylPhenylalanine;4MethylPhealanine; Homophenylalanine (hPhe); 4-aminophenylalanine(Phe4NH(Boc); phenyl glycine; Pipecolic acid (Pip); propargylglycine;Thioproline (Thz); ButylGlycine (Tle); 3-NitroTyrosine.

As used herein, the term “residue” when referring to α-amino acids isintended to mean a radical derived from the corresponding α-amino acidby eliminating the hydroxyl of the carboxy group and one hydrogen of theα-amino group. For example, the terms Gln, Ala, Gly, Ile, Arg, Asp, Phe,Ser, Leu, Cys, Asn, and Tyr represent the residues of L-glutamine,L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid,L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine, andL-tyrosine, respectively.

As used herein the term “amino acid side chain” is intended to mean thepart of an amino acid's chemistry that differentiates it from otheramino acids. Amino acid structure includes a carboxyl group, an aminegroup plus the individual side chain. Each amino acid has a unique sidechain. This is applied to unnatural amino acids as well. This side chainmay exist in protected form or not.

As used herein, the term “prodrug” is intended to mean a compound thatmay be converted under physiological conditions or by solvolysis to abiologically active compound of the present invention. Thus, the term“prodrug” refers to a precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive or displaylimited activity when administered to a subject in need thereof, but isconverted in vivo to an active compound of the present invention.Typically, prodrugs are transformed in vivo to yield the compound of theinvention, for example, by hydrolysis in blood or other organs byenzymatic processing. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in the subject (see,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). The definition of prodrug includes any covalently bondedcarriers which release the active compound of the invention in vivo whensuch prodrug is administered to a subject. Prodrugs of a compound of thepresent invention may be prepared by modifying functional groups presentin the compound of the invention in such a way that the modificationsare cleaved, either in routine manipulation or in vivo, to a parentcompound of the invention.

As used herein, the term “pharmaceutically acceptable salt” is intendedto mean both acid and base addition salts.

As used herein, the term “pharmaceutically acceptable acid additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free bases, which are notbiologically or otherwise undesirable, and which are formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, trifluoroacetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like.

As used herein, the term “pharmaceutically acceptable base additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.

The compounds of the present invention, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers, chiral axesand chiral planes and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms and may be defined in terms of absolutestereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for aminoacids. The present invention is intended to include all such possibleisomers, as well as, their racemic and optically pure forms. Opticallyactive (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques, such as reverse phase HPLC. The racemicmixtures may be prepared and thereafter separated into individualoptical isomers or these optical isomers may be prepared by chiralsynthesis. The enantiomers may be resolved by methods known to thoseskilled in the art, for example by formation of diastereoisomeric saltswhich may then be separated by crystallization, gas-liquid or liquidchromatography, selective reaction of one enantiomer with an enantiomerspecific reagent. It will also be appreciated by those skilled in theart that where the desired enantiomer is converted into another chemicalentity by a separation technique, an additional step is then required toform the desired enantiomeric form. Alternatively specific enantiomersmay be synthesized by asymmetric synthesis using optically activereagents, substrates, catalysts, or solvents or by converting oneenantiomer to another by asymmetric transformation.

Certain compounds of the present invention may exist as a mix ofepimers. Epimers means diastereoisomers that have the oppositeconfiguration at only one of two or more stereogenic centres present inthe respective compound.

Certain compounds of the present invention may exist in Zwitterionicform and the present invention includes Zwitterionic forms of thesecompounds and mixtures thereof.

In addition, the compounds of the invention also may exist in hydratedand anhydrous forms. Hydrates of the compound of any of the formulasdescribed herein are included as compounds of the invention. In afurther embodiment, the compound according to any of the formulasdescribed herein is a monohydrate. In one embodiment, the compound ofthe invention comprises about 10% or less, about 9% or less, about 8% orless, about 7% or less, about 6% or less, about 5% or less, about 4% orless, about 3% or less, about 2% or less, about 1% or less, about 0.5%or less, about 0.1% or less by weight of water. In another embodiment,the compounds of the invention comprise, about 0.1% or more, about 0.5%or more, about 1% or more, about 2% or more, about 3% or more, about 4%or more, about 5% or more, or about 6% or more by weight of water.

C) Methods of Preparation

General methods for the synthesis of the compounds of the presentinvention are shown below and are disclosed merely for the purpose ofillustration and are not meant to be interpreted as limiting theprocesses to make the compounds by any other methods.

Those skilled in the art will readily appreciate that a number ofmethods are available for the preparation of the compounds of thepresent invention.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Sigma-AldrichChemicals, Anaspec or chemipex.

The starting materials and the intermediates of the reaction may beisolated and purified if desired using conventional techniques suchchromatography (Biotage flash chromatography), filtration, distillation,etc. Such materials can be characterized using conventional analyticalmethods such as NMR and LCMS.

The coupling step is carried out in the presence of a suitable couplingagent such as, but not limited to, diisopropyl carbodiimide (DPC),anhydride mixte (isobutylchloroformate),O-benzotriazole-1-yloxytrispyrrolidinophosphoniumhexafluorophosphate(PyBOP), O-benzotriazol-1-yl-N,N,N,Ntetramethyluroniumhexafluoro-phosphate (HBTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (HATU), 1-hydroxybenzotriazole (HOBT) in thepresence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC). A base such as N,N-diisopropylethylamine, triethyl amine, orN-methylmorpholine. The reaction is carried out at 20° C. except foranhydride mix formation (isobutyl chloroformate, −5/−13° C.) to avoidany racemisation of amino acids.

Removal of the amino protecting group is carried out but by piperidinein dichloromethane (Fmoc protecting group). The removal of β-tert-butylcarboxylic acid is carried out by TFA. Selective removal of N-Boc fromB-tert butyl aspartic acid is carried out by PTSA or TFA at 0° C.

Vinyl sulfone function is elucidated as an example only in the followingexamples, but is not limited to it. It will be recognized by a skilledperson in the art, that other vinyl electron-withdrawing group (EWG) maybe used in this invention. Benzyloxycarbonyl (Z) is elucidated as anexample of linkers that may facilitate the penetration of the drug intocells. It is recognized that other X—R3 may be used as well. The allylgroup was introduced directly by the use of commercially availableAA₂-OAllyl protected form or synthesized from the corresponding aminoacid AA₂-OH.

This is a convergent synthesis, which consists of synthesizing twodifferent fragments (suicide inhibitor linker and the peptide orpeptidomimetic fragment) prior to the ‘coupling step’.

A Compound of Formula 1a can be Prepared by the Procedure DescribedBelow

Left Arm Synthesis:

The allyl group was introduced directly by the use of commerciallyavailable AA₂-OAllyl protected form or synthesized from the protectedamino acid fmoc-AA₂-OH and allyl alcohol (EDC, DMAPcat, NMM, CH₂Cl₂/DMF(5/1)) followed with fmoc deprotection (piperidine, CH₂Cl₂).

The AA₂-OAllyl is coupled to Fmoc-AA₃-OH with the coupling reagent1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) andsubsequent fmoc deprotection with piperidine gives AA₃-AA₂-Oallyl(intermediate A) ready for coupling with (Z) AA₄-OH gave (Z)AA₄-AA₃-AA₂-Oallyl (intermediate B) which upon removal of allyl group(tetrakis) liberate the C-terminal carboxylic acid (tripeptide A) asdescribed in the following schem.

Right Arm Synthesis

The suicide substrate proposed in the following scheme is Aspα-chlorovinyl methylsulfone.

The common intermediate Boc-Asp(B-tert-butyl)-His synthesized fromBoc-Asp(B-tert-butyl)-N-hydroxysuccinimide ester as reported by (MancusoA et al., 1981, William R. Ewing et al., 1999 and Won Bum Jang. 2004).

Treatment of the aldehyde with sodium anion of Diethylchloro(methylsulfone)methylphosphonate results in the correspondingBoc-Asp β-tert-butyl) α-chlorovinyl-methylsulfone in the manner ofWadsworth and Emmons.

Coupling Step Synthesis

The coupling step between Asp a chlorovinyl methylsulfone salt and thepeptide in the presence of coupling reagents such (isobutylchloroformate, NMM) or (HOBT, NMM, EDC; Dragovich P., S., 1999) resultsin Asp α-chlorovinyl methylsulfone peptide derivative.

A Compound of Formula 1b can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following scheme is Aspα-chlorovinyl phenylsulfone.

Treatment of the aldehyde with sodium anion of Diethylchloro(methylsulfone)phenyl phosphonate results in the correspondingBoc-Asp (β-tert-butyl) α-chlorovinyl phenylsulfone in the manner ofWadsworth and Emmons.

Coupling Step Synthesis

The coupling step between Asp α-chlorovinyl phenylsulfone salt and thepeptide in the presence of coupling reagents such (isobutylchloroformate, NMM) or (HOBT, NMM, EDC) results in Asp α-chlorovinylphenylsulfone peptide derivative.

A Compound of Formula 1c can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following schem is Aspα-methoxyvinyl methylsulfone.

Oxydation of diethyl methylthiomethylphosphonate include anodicoxidation in NaOMe/MeOH (saturated with CO2) to give the O, S-acetal ofdiethoxyphosphinylformaldehyde, which upon oxidation may give thecorresponding sulfone as Diethyl methoxy(methylsulfone)phenylphosphonate (Shankaran, K et al. 2001).

Treatment of the aldehyde with sodium anion of Diethyl methoxy(methylsulfone)phenyl phosphonate should result in the correspondingBoc-Asp (β-tert-butyl) α-methoxyvinyl methylsulfone in the manner ofWadsworth and Emmons.

Coupling Step Synthesis

The coupling step between Asp α-methoxyvinyl methylsulfone salt and thepeptide in the presence of coupling reagents such (isobutylchloroformate, NMM) or (HOBT, NMM, EDC) results in Asp α-methoxyvinylmethylsulfone peptide derivative.

A Compound of Formula 1d can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following schem isAsp(B-Methyl)methyl vinylsulfone.

Boc-Asp(B-Methyl)-N-hydroxysuccinimide ester was reduced to thecorresponding alcohol (NaBH₄, THF), a subsequent oxidation (oxalylchloride, DMSO) gave the aldehyde: Boc-Asp(B-Methyl)-H.

Treatment of Boc-Asp(B-Methyl)-H with sodium anion of Diethyl(methylsulfone) methyl phosphonate results in the corresponding Boc-Asp(β-Methyl)methyl vinylsulfone in the manner of Wadsworth and Emmons.

Coupling Step Synthesis

The coupling step between Asp (β-Methyl)methyl vinylsulfone salt and thepeptide in the presence of coupling reagents such (isobutylchloroformate, NMM) or (HOBT, NMM, EDC) results in Asp (β-Methyl)methylvinylsulfone peptide derivative.

A Compound of Formula 1e can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following schem is Asp methylvinyllsulfone.

The common intermediate Boc-Asp(B-tert-butyl)-His synthesized fromBoc-Asp(B-tert-butyl)-N-hydroxysuccinimide ester as reported by (MancusoA et al., 1981, William R. Ewing et al., 1999 and Won Bum Jang. 2004).

Treatment of the aldehyde with sodium anion of Diethyl (methylsulfone)methyl phosphonate results in the corresponding Boc-Asp(β-tert-butyl)methylvinylsulfone in the manner of Wadsworth and Emmons,1961 and Palmer et al. 1995.

Coupling Step Synthesis

The coupling step between Asp methyl vinylsulfone salt and the peptidein the presence of coupling reagents such (isobutyl chloroformate, NMM)or (HOBT, NMM, EDC) results in Asp methyl vinylsulfone peptidederivative.

A Compound of Formula 1f can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following schem is Asp phenylvinyllsulfone.

Diethyl phenylsulfonylmethylphosphonate was obtained in one step frombenzenesulfonyl fluoride and triethyl phosphorane in the presence oflithium hexamethyldisilazide (Won Bum Jang et al., 1998).

Treatment of the aldehyde with sodium anion of Diethyl (phenyl sulfone)methylphosphonate results in the corresponding Boc-Asp(β-tert-butyl)phenyl vinylsulfone in the manner of Wadsworth and Emmons,1961 and Palmer et al. 1995.

Coupling Step Synthesis

The coupling step between Asp phenyl vinylsulfone salt and the peptidein the presence of coupling reagents such (isobutyl chloroformate, NMM)or (HOBT, NMM, EDC) results in Asp phenyl vinylsulfone peptidederivative.

A Compound of Formula 1g can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following schem is Asp phenoxyvinyllsulfone.

Diethyl (phenoxysulfone) methylphosphonate was obtained frommethanesulfonyl phenoxy and diethyl chlorophosphonate in the presence ofpotassium bis(trimethylsilyl)amide. A subsequent oxidation (AcOH, H₂O₂)gave the corresponding sulfone.

Treatment of the aldehyde with sodium anion of Diethyl (phenoxy sulfone)methylphosphonate results in the corresponding Boc-Asp(β-tert-butyl)phenoxy vinylsulfone in the manner of Wadsworth andEmmons.

Coupling Step Synthesis

The coupling step between Asp phenoxy vinylsulfone salt and the peptidein the presence of coupling reagents such (isobutyl chloroformate, NMM)or (HOBT, NMM, EDC) results in Asp phenoxy vinylsulfone peptidederivative.

A Compound of Formula 1h can be Prepared by the Procedure DescribedBelow

Right Arm Synthesis

The suicide substrate proposed in the following schem is Asp morpholinevinyllsulfone.

Diethyl (morpholinesulfone) methylphosphonate was prepared from methanesulfonyl morpholine and chloromethylphosphonate in the presence ofpotassium bis(trimethylsilyl) amide.

Treatment of the aldehyde with sodium anion of Diethyl(morpholinesulfone) methylphosphonate results in the correspondingBoc-Asp (β-tert-butyl)morpholino vinylsulfone in the manner of Wadsworthand Emmons.

Coupling Step Synthesis

The coupling step between Asp morpholine vinylsulfone salt and thepeptide in the presence of coupling reagents such (isobutylchloroformate, NMM) or (HOBT, NMM, EDC) results in Asp morpholinevinylsulfone peptide derivative

A Compound of Formula 1i can be Prepared by the Procedure Below

Right Arm Synthesis

The suicide substrate proposed in the following schem isAsp(B-Methyl)-vinylsulfone derivatives.

Boc-Asp(B-Methyl)-N-hydroxysuccinimide ester was reduced to thecorresponding alcohol (NaBH₄, THF), a subsequent oxidation (oxalylchloride, DMSO) gave the aldehyde Boc-Asp(B-Methyl)-H.

Treatment of Boc-Asp(B-Methyl)-H with sodium anion of Diethyl(methylsulfone) phosphonate derivatives results in the correspondingBoc-Asp (β-Methyl) vinylsulfone derivatives in the manner of Wadsworthand Emmons.

Coupling Step Synthesis

The coupling step between Asp (β-Methyl) vinylsulfone derivatives saltand the caspase-3 designed peptide in the presence of coupling reagentssuch (isobutyl chloroformate, NMM) or (HOBT, NMM, EDC) results inZ-Asp(β-Methyl)-AA₃-AA₂-Asp (β-Methyl) vinylsulfone peptide derivatives.

This approach will allow the synthesis of a variety of pro-drugvinylsulfone derivatives that could be easily obtained by choosing theappropriate combination (X, R) and applying the appropriate methodmentioned above. Such combination may enhance cell permeability,selectivity and potency.

All acid, salt and other ionic and non-ionic forms of the compoundsdescribed are included as compounds of the invention. For example, if acompound is shown as an acid herein, the salt forms of the compound arealso included. Likewise, if a compound is shown as a salt and the acidforms are also included.

In certain embodiments, the compounds of the present invention arerepresented by generalized Formula I, IA, II, IIA, III, or IIIA, or apharmaceutically acceptable salt and/or prodrug thereof:

D) Development of Specific Caspase Inhibitors

An additional aspect of the invention relates to a method for designingcaspase inhibitors. Following the same approach as outlined hereinafter,those skilled in the art will appreciate that it is conceivable tofurther improve the potency of the compounds of Formula II, e.g.Z-Asp-Phg-Val-AspVSmethyl (53), against caspase-3 and inhibitselectively additional caspases such as caspase-2, caspase-8, caspase-9and caspase-1.

As is known, all caspases cleave substrates to the right of the asparticacid amino acid in position P1. However, caspase-3 requires anadditional Asp at position P4, which confers caspase-3 its specificity:

As shown in Table 2 hereinafter, suicide substrates such as AspVSmethyl(Compound 93) and Asp(Otbu)VSmethyl (Compound 33) are devoid of anyactivity against caspase-3. The substrate z-Asp(Otbu)-Phg-val-OH(Compound 16) is also devoid of any activity against caspase-3. Howeverthe fusion product of peptide and suicide substrate,z-Asp-Phg-Val-AspVSmethyl (Compound 53), proved to be a very potentinhibitor of caspase-3 with an IC50 30-90 nM. Inhibition is selectivesince even though caspase-7 belongs to the same group than caspase-3,the IC50 value for this caspase was about 12 fold higher. Replacement ofPhg at position P3 with Ala (2′-quinolyl) enhanced the selectivityfurther (about 56 fold difference, see Compound 55).

As observed with z-Val-AspVSmethyl (Compound 61), the deletion of bothAspartic acid and Ala(2′-quinolyl) at P4 and P3 positions abolishedcompletely the activity against caspase-3. The same result was observedafter the deletion of Aspartic acid only in the example of z-Ala(2′-quinolyl)-Val-Asp alpha chlorovinyl methyl sulfone (Compound 50)compared with z-Asp-Ala(2′-quinolyl)-Val-Asp alpha chlorovinyl methylsulfone (Compound 48).

Changes at Position P3 and P2

The amino acids at both position P3 and P2 can serve to selectivelytarget caspase-3 as observed but also to selectively target othercaspases. The following examples highlight this possibility: (1) Thereplacement of Ala(2′-quinolyl) (Compound 55) with indalylglycine at P3position lead to z-Asp-indalylglycine-Val-AspVSmethyl (Compound 57).This substitution enhanced the inhibitory effect against the group IIIof caspases (caspase-3 (30-90 nM) and 7 (0.18-0.30 uM) and, with about29 fold less efficiency, against group 1 (caspase-1 (0.6-1.2 uM)). (2)The presence of Trp at P3 position (z-Asp-Trp-Val-AspVSmethyl; Compound88) retained the selectivity against caspase-3 (30-90 nM) and producedan additional activity against caspase-1 (0.6-1.2 uM). This moleculepossesses therefore the capability to inhibit selectively two caspasesbelonging to two different groups, namely the proinflammatory and theproapoptotic group. (3) The presence of Glutamic acid at P-3 position(z-Asp-Glu-Val-AspVSmethyl; Compound 59) retained the selectivityagainst caspase-3 (IC50 20 nM) and produced an additional activityagainst caspase-7 (IC50 42 nM) and caspase-9 (509 nM). This moleculetherefore possesses the capability to inhibit selectively two caspasesbelonging to two different groups, namely initiator and executionercaspases.

Changes in Position P4

Changes in position P4 can also affect the selectivity of a giveninhibitor. The amino acid that has been showed to fit well into thecorresponding caspase-1 pockets at position P4 is Tyrosine. Therefore,z-Tyr-Val-Ala-AspVS phenyl (Compound 96) was tested against differentcaspases, and it proved to be selective against caspase-1 (IC50 1.2-1.5μM). The amino acid that has been showed to fit well into thecorresponding caspase-1 pockets at position P3 is glutamic acid.Therefore, z-Tyr-Glu-Ala-AspVS methyl (Compound 82) was tested againstdifferent caspases and the inhibition of caspase-1 was enhanced to 0.5μM.

These specific examples demonstrate that it is possible to makeselective caspase-3 inhibitors based on sequences recognized by groupIII caspases. Following the same approach as outlined hereinafter, it isconceivable to inhibit selectively additional caspases and to furtherimprove the potency against selected caspases.

Design for Selective Caspase-3 Inhibitors

In one embodiment, the method comprises synthesizing compounds havingthe following general Formula D1:

D_(a)-(P3)-(P2)-D_(b)-suicide substrate  (D1)

whereinP2 is selected from the following amino acids: V, L, P, M, A, T, and H;P3 is selected from the following amino acids: Phg, E, Indanylglycine,W, Y, A, D, Ala-(2′-quinolyl), Q, F, S, T, V, Y, G, L;D_(a) is (D) or (L) aspartic acid.D_(b) is the side chain of (D) or (L) aspartic acid.

The following compound (DEVD-vinyl phenyl sulfone) is an example acompounds having a sequence (i.e. D_(a) is Cbz-aspartic acid; P3 is Glu;P2 is Val; D_(b) is Asp; and the suicide substrate is vinyl phenylsulfone) designed to selectively inhibit caspase-3:

Design for Selective Caspase-8/Caspase-9 Inhibitors

In one embodiment, the method comprises synthesizing compounds havingthe following general Formula D2:

(P4)-(P3)-(P2)-D-suicide substrate  (D2)

whereP2 is selected from the following amino acids: T, H, V, W, I, and A;P3 is selected from the following amino acids: E, Ala(2′-quinolyl;P4 is selected from the following amino acids: I, L, E, D, A, P, and V;D is the side chain of (D) or (L) aspartic acid and the suicidesubstrate is vinyl phenyl sulfone.

The following is an example of a compound having a sequence (i.e. P4 isCbz-L; P3 is Glu; P2 is His; D is Asp and the suicide substrate is vinylphenyl sulfone) designed to selectively inhibit caspase-8:

Design for Selective Caspase-2 Inhibitors

In one embodiment, the method comprises synthesizing compounds havingthe following general Formula D3:

(P5)-(P4)-(P3)-(P2)-D-suicide substrate  (D3)

whereP2 is selected from the following amino acids: A, S, K and V;P3 is selected from the following amino acids: V, E, T and Q;P4 is selected from the following amino acids: D and L;P5 is selected from the following amino acids: V and L;D is the side chain of (D) or (L) aspartic acid; andthe suicide substrate is selected from the group consisting of vinylphenyl sulfone.

The following is an example of a compound (VDEHD-vinylphenyl sulfone)having a sequence (i.e. P5 is Cbz Val; P4 is Asp; P3 is Glu; P2 is Hisand D is Asp. The suicide substrate is vinyl phenyl sulfone), which isdesigned to selectively inhibit caspase-2:

Design for Selective Caspase-1 Inhibitors

In one embodiment, the method comprises synthesizing compounds havingthe following general Formula D4:

(P4)-(P3)-(P2)-D-suicide substrate  (D4)

whereP2 is selected from the following amino acids: V, A, T, and H.P3 is selected from the following amino acids: E, Q, D, A, G, T, V,Ala(2′-quinolyl), indanylglycine, and W;P4 is selected from the following amino acids: Y, W, F, and D;D is the side chain of (D) or (L) aspartic acid; andthe suicide substrate is selected from the group consisting of vinylphenyl sulfone.

The following is an example of a compound (YEHD-vinylphenyl sulfone)having a sequence (i.e. P4 is Cbz Tyr; P3 is Glu; P2 is H is; D is Aspand the suicide substrate is vinyl phenyl sulfone, which is designed toselectively inhibit caspase-1:

D) Pharmaceutical Applications

As indicated hereinbefore and exemplified hereinafter, the compounds ofthe invention have beneficial pharmaceutical properties and thesecompounds may have pharmaceutical applications in the prevention and/ortreatment of various diseases and conditions in a subject. Medical andpharmaceutical applications contemplated by the inventors include, butare not limited to, caspase-mediated diseases. In addition, thecompounds of the present invention may have useful benefits on cells invitro such as promoting cell survival or the health of the cells.

The term “subject” includes living organisms in which blood disorders,renal failure, inflammatory-related diseases associated with high bloodpressure, and/or oxidative stress-related disorders, can occur, or whichare susceptible to such conditions. The term “subject” includes animals(e.g., mammals (e.g., cats, dogs, horses, pigs, cows, goats, sheep,rodents (e.g., mice or rats), rabbits, squirrels, bears, primates (e.g.,chimpanzees, monkeys, gorillas, and humans)), as well as avians (e.g.chickens, ducks, Peking ducks, geese), and transgenic species thereof.Preferably, the subject is a mammal. More preferably, the subject is ahuman. Even more preferably, the subject is a human patient in need oftreatment.

The term “caspase-mediated disease” includes all diseases, disorderand/or conditions in which any one or more of caspase-1, -2, -3, -4, -5,-6, -7, -8, -9, -10, -11, -12, plays a significant role. In someembodiments, the caspase-mediated disease mainly involves executionercaspases (caspase-3, 6, 7). In another embodiment, the caspase-mediateddisease mainly involves initiators (caspase-2, 8, 9, 10). In someembodiments, a compound of the invention shows a high specificitytowards one particular caspase. In another embodiment, a compound of theinvention is able to inhibit two groups of caspases. Yet, in anotherembodiment, a compound of the invention even is able to inhibit twospecific caspases belonging to two different groups of caspases.

Examples of caspase-mediated disease according to the inventionincludes, but are not limited to, apoptosis mediated diseases, IL-1mediated diseases, inflammatory diseases, autoimmune diseases,autoinflammatory diseases, proliferative diseases, infectious diseases,degenerative diseases, retinal disorders, inflammatory peritonitis,osteoarthritis, pancreatitis, asthma, respiratory distress syndrome,rheumatoid arthritis, systemic lupus erythematous, scleroderma, Grave'sdisease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia,autoimmune neutropenia, thrombocytopenia, hepatitis, inflammatory boweldisease, crohn's disease, psoriasis, dermatitis, Graft vs host disease,organ transplant rejection, osteoporosis, leukemias and relateddisorders, multiple myeloma-related diseases, metastatic melanomas,Kaposi's sarcoma, sepsis, septic shock, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, cerebral ischemia, epilepsy, myocardialischemia, acute and chronic heart disease, myocardial infarction,congestive heart failure, atherosclerosis, spinal muscular atrophy,amyotrophic lateral sclerosis, multiple sclerosis, HIV-relatedencephalitis, aging, neurological damage due to stroke, ulcerativecolitis, traumatic brain injury, spinal cord injury, hepatitis-B,hepatitis-C, hepatitis-G, liver-related diseases, renal disease, and HIVinfection.

As used herein, “preventing” or “prevention” is intended to refer to atleast the reduction of likelihood of the risk of (or susceptibility to)acquiring a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a patient that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease). Biological and physiologicalparameters for identifying such patients are provided herein and arealso well known by physicians.

The terms “treatment” or “treating” of a subject includes theapplication or administration of a compound of the invention to asubject (or application or administration of a compound of the inventionto a cell or tissue from a subject) with the purpose of stabilizing,curing, healing, alleviating, relieving, altering, remedying, lessworsening, ameliorating, improving, or affecting the disease orcondition, the symptom of the disease or condition, or the risk of (orsusceptibility to) the disease or condition. The term “treating” refersto any indicia of success in the treatment or amelioration of an injury,pathology or condition, including any objective or subjective parametersuch as abatement; remission; lessening of the rate of worsening;lessening severity of the disease; stabilization, diminishing ofsymptoms or making the injury, pathology or condition more tolerable tothe subject; slowing in the rate of degeneration or decline; making thefinal point of degeneration less debilitating; or improving a subject'sphysical or mental well-being. In some embodiments, the term “treating”can include increasing a subject's life expectancy and/or delay beforeadditional treatments are required.

Addressing caspase-mediated diseases is among the medical andpharmaceutical applications contemplated by present invention.Therefore, in one of its aspects the present invention relates tomethods, compounds and compositions for prevention and/or treatment of acaspase-mediated disease in a subject, preferably a human patient inneed thereof.

Another aspect of the invention relates to the use of the compoundsdescribed herein for inhibiting a caspase or a caspase-like protein in acell, comprising contacting the caspase or caspase-like protein with aneffective amount of a caspase inhibitor according to the invention.

In some embodiments, the subject may be suffering from a viralinfection. Therefore, the invention also relates to a method for theprophylaxis or therapy of a viral infection, comprising administering toa subject in need thereof an effective dose of a caspase inhibitoraccording to the invention (or a pharmaceutical composition comprisingthe same). This may be helpful for inhibiting a cellular caspase therebyinhibiting virus multiplication.

Also of particular interest is a method for the treatment of excessiveapoptosis affected by caspase activity in a cell or a tissue, comprisingcontacting the cell or the tissue with an effective amount of one ormore caspase inhibitor according to the invention (or a pharmaceuticalcomposition comprising the same).

Also of particular interest is a method for simulating stem cellproliferation by preventing some of the stem cells from entering apartial or complete apoptosis cycle. The method for culturing a largequantity of stem cells may involves an effective amount of one or morecaspase inhibitor according to the invention (or a pharmaceuticalcomposition comprising the same) and a medium for culturing stem cells.

Although focusing on caspases, the present is not so limited. Forinstance, it is conceivable that the compounds of the invention be alsoeffective in inhibiting additional families of proteases, including butnot limited to, serine peptidases, cysteine peptidases, asparticpeptidases, metallo-peptidases, and other peptidases of unknowncatalytic type. For a more elaborate listing of proteases that may beinhibited by the compounds defined herein, see ZBIGNIEW GRZONKA.Cysteine protease. Industrial Enzymes, 181-195, Chapter 11, 2007Springer.

In order to evaluate, assess, and/or confirm the efficacy of the method,compounds and/or compositions of the invention, serial measurements canbe determined. Quantitative assessment of caspase functions andparameters of caspase dysfunction are well known in the art. Examples ofassays for the determination of caspases activity are provided in theExemplification section.

The compounds according to the invention can be further analyzed, testedor validated for their ability to cross the Blood Brain Barrier BBB isso desired. Many in-vitro, in-vivo and in-silico methods may be employedduring drug development to mimic the BBB (Lohmann et al. (2002)Predicting blood-brain barrier permeability of drugs: evaluation ofdifferent in vitro assays. J Drug Target 10:263-276; Nicolazzo et al.(2006) Methods to assess drug permeability across the blood-brainbarrier. J Pharm Pharmacol 58:281-293). In-vitro models include primaryendothelial cell culture and immortalized cell lines such as Caco-2,BMEC, MDCK. These cells are useful as a screening method and canappropriately rank compounds in order of BBB permeability. In vivomodels such as the internal carotid artery single injection orperfusion, intravenous bolus injection, brain efflux index andintracerebral microdialysis provide more accurate information regardingbrain uptake, and these can be complemented with novel imagingtechniques (such as magnetic resonance imaging and positron emissiontomography), although such methods are not suited to high-throughputpermeability assessment.

In certain embodiments, at least some of the prodrugs administeredgenerates the corresponding pharmaceutical compound only afterabsorption by the gastrointestinal tract and/or only once it has reachedthe brain, i.e. after it has passed the blood brain barrier (BBB).

E) Pharmaceutical Compositions and Formulations

A related aspect of the invention concerns pharmaceutical compositionscomprising one or more of the compounds of the invention describedherein. As indicated hereinbefore, the compounds of the invention may beuseful in preventing and/or treating caspase-mediated disease, and moreparticularly sepsis, myocardial infarction, ischemic stroke, spinal cordinjury (SCI), traumatic brain injury (TBI) and neurodegenerative disease(e.g. multiple sclerosis (MS) and Alzheimer's, Parkinson's, andHuntington's diseases.

As used herein, the term “therapeutically effective amount” means theamount of compound that, when administered to a subject for treating orpreventing a particular disorder, disease or condition, is sufficient toeffect such treatment or prevention of that disorder, disease orcondition. Dosages and therapeutically effective amounts may vary forexample, depending upon a variety of factors including the activity ofthe specific agent employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, and any drug combination, ifapplicable, the effect which the practitioner desires the compound tohave upon the subject and the properties of the compounds (e.g.bioavailability, stability, potency, toxicity, etc), and the particulardisorder(s) the subject is suffering from. In addition, thetherapeutically effective amount may depend on the subject's bloodparameters (e.g. lipid profile, insulin levels, glycemia), the severityof the disease state, organ function, or underlying disease orcomplications. Such appropriate doses may be determined using anyavailable assays including the assays described herein. When one or moreof the compounds of the invention is to be administered to humans, aphysician may for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained.

As used herein, the term “pharmaceutical composition” refers to thepresence of at least one compound of the invention according to any oneof Formula I, IA, II, IIA, III, or IIIA as defined herein and at leastone pharmaceutically acceptable vehicle. Examples of representativecompounds of the invention include the compounds in Table 1 andpharmaceutically acceptable salts thereof.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which a compound is administered. The term“pharmaceutically acceptable” refers to drugs, medicaments, inertingredients etc., which are suitable for use in contact with the tissuesof humans and lower animals without undue toxicity, incompatibility,instability, irritation, allergic response, and the like, commensuratewith a reasonable benefit/risk ratio. It preferably refers to a compoundor composition that is approved or approvable by a regulatory agency ofthe Federal or state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for use in animals and moreparticularly in humans. The pharmaceutically acceptable vehicle can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.Additional examples of pharmaceutically acceptable vehicles include, butare not limited to: Water for Injection USP; aqueous vehicles such as,but not limited to, Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, and LactatedRinger's Injection; water-miscible vehicles such as, but not limited to,ethyl alcohol, polyethylene glycol, and polypropylene glycol; andnon-aqueous vehicles such as, but not limited to, corn oil, cottonseedoil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, andbenzyl benzoate. Prevention of the action of microorganisms can beachieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, isotonic agents are included, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.Nanoparticles, liposomes, and antibodies conjugated to nanoparticles orcombinations thereof, are also contemplated as pharmaceuticallyacceptable vehicles.

In some embodiments, the compositions of the invention comprise aneffective amount of a compound of the Formula I, IA, II, IIA, III, orIIIA as described hereinbefore, preferably compoundZ-Asp-Indanylglycine-Val-Aspmethyl vinyl sulfone (57);Z-Asp-Glu-Val-Aspmethyl vinyl sulfone (59)Z-Asp-Ala(2′-quinolyl)-Val-Aspmethyl vinyl sulfone (55);Z-Asp-Phg-Val-Aspmethyl vinyl sulfone (53);Z-Asp-Ala(2′-quinolyl)-Val-Asp-αchlorovinyl methylsulfone (48);Z-Asp(β-methyl)-Indanylglycine-Val-Asp(β-methyl)methyl vinyl sulfone(51) Z-Asp-Tyr-Val-Aspmethyl vinyl sulfone (76) Z-Asp-Trp-Val-Aspmethylvinyl sulfone (88) or a pharmaceutically acceptable salt thereof.

In some embodiments the invention pertains to pharmaceuticalcompositions for preventing and/or treating diseases or other medicalconditions in which at least one caspase is significantly involved thatinclude one or more compounds of Formula I, IA, II, IIA, III, or IIIA asdefined herein.

In some embodiments the invention pertains to pharmaceuticalcompositions for preventing and/or treating diseases or other medicalconditions in which at least one caspase is significantly involved, thecomposition comprising one or more compounds of Formula I, IA, II, IIA,III, or IIIA as defined herein.

The compounds of the invention may be formulated prior to administrationinto pharmaceutical compositions using available techniques andprocedures. For instance, the pharmaceutical compositions may beformulated into suitable administration (orally, parenterally,(intravascular (IV), intraarterial (IA), intramuscular (IM), depo-IM,subcutaneous (SC), and depo SC), sublingually, intranasally(inhalation), intrathecally, topically, or rectally.

Preferably, the compound(s) of the invention can be orally administered.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy.Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention with apharmaceutically acceptable vehicle (e.g. an inert diluent or anassimilable edible carrier) and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product. The amount of the therapeuticagent in such therapeutically useful compositions is such that asuitable dosage will be obtained.

Formulations of the invention suitable for oral administration may be inthe form of capsules (e.g. hard or soft shell gelatin capsule), cachets,pills, tablets, lozenges, powders, granules, pellets, dragees, e.g.,coated (e.g., enteric coated) or uncoated, or as a solution or asuspension in an aqueous or non-aqueous liquid, or as an oil-in-water orwater-in-oil liquid emulsion, or as an elixir or syrup, or as pastillesor as mouth washes and the like, each containing a predetermined amountof a compound of the present invention as an active ingredient. Acompound of the present invention may also be administered as a bolus,electuary or paste, or incorporated directly into the subject's diet.Moreover, in certain embodiments these pellets can be formulated to (a)provide for instant or rapid drug release (i.e., have no coating onthem); (b) be coated, e.g., to provide for sustained drug release overtime; or (c) be coated with an enteric coating for bettergastrointestinal tolerability. Coating may be achieved by conventionalmethods, typically with pH or time-dependent coatings, such that thecompound(s) of the invention is released in the vicinity of the desiredlocation, or at various times to extend the desired action. Such dosageforms typically include, but are not limited to, one or more ofcellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropylmethyl cellulose phthalate, ethyl cellulose, waxes, and shellac.

In solid dosage forms for oral administration a compound of the presentinvention may be mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, or any of thefollowing: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose or acacia; humectants, such as glycerol; disintegrating agents,such as agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain silicates, and sodium carbonate; solution retardingagents, such as paraffin; absorption accelerators, such as quaternaryammonium compounds; wetting agents, such as, for example, cetyl alcoholand glycerol monostearate; absorbents, such as kaolin and bentoniteclay; lubricants, such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof;and coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

Peroral compositions typically include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically acceptable vehiclessuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, tragacanth, and sodium alginate; typical wetting agentsinclude lecithin and polysorbate 80; and typical preservatives includemethyl paraben and sodium benzoate. Peroral liquid compositions may alsocontain one or more components such as sweeteners, flavoring agents andcolorants disclosed above.

Pharmaceutical compositions suitable for injectable use may includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. Sterile injectable solutions can be prepared byincorporating the therapeutic agent in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the therapeutic agent into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, themethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient (i.e., the therapeutic agent) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired compound of any Formula hereinor a plurality of solid particles of such compound(s). The desiredformulation may be placed in a small chamber and nebulized. Nebulizationmay be accomplished by compressed air or by ultrasonic energy to form aplurality of liquid droplets or solid particles comprising the agents orsalts. The liquid droplets or solid particles should have a particlesize in the range of about 0.5 to about 5 microns. The solid particlescan be obtained by processing the solid agent of any Formula describedherein, or a salt thereof, in any appropriate manner known in the art,such as by micronization. The size of the solid particles or dropletswill be, for example, from about 1 to about 2 microns. In this respect,commercial nebulizers are available to achieve this purpose. Apharmaceutical formulation suitable for administration as an aerosol maybe in the form of a liquid, the formulation will comprise awater-soluble agent of any Formula described herein, or a salt thereof,in a carrier which comprises water. A surfactant may be present whichlowers the surface tension of the formulation sufficiently to result inthe formation of droplets within the desired size range when subjectedto nebulization.

The compositions of this invention can also be administered topically toa subject, e.g., by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch”. Such compositions include, for example, lotions, creams,solutions, gels and solids. These topical compositions may comprise aneffective amount, usually at least about 0.1%, or even from about 1% toabout 5%, of a compound of the invention. Suitable carriers for topicaladministration typically remain in place on the skin as a continuousfilm, and resist being removed by perspiration or immersion in water.Generally, the carrier is organic in nature and capable of havingdispersed or dissolved therein the therapeutic agent. The carrier mayinclude pharmaceutically acceptable emollients, emulsifiers, thickeningagents, solvents and the like.

Other compositions useful for attaining systemic delivery of the subjectagents include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

The compound(s) of the invention may also be administered parenterally,intraperitoneally, intravenously, intraspinally, intrathecally orintracerebrally. For such compositions, the compound(s) of the inventioncan be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

The method of treatment of the present invention may also includeco-administration of the at least one compound according to theinvention, or a pharmaceutically acceptable salt thereof together withthe administration of another therapeutically effective agent.Therefore, an additional aspect of the invention relates to methods ofconcomitant therapeutic treatment of a subject, comprising administeringto a subject in need thereof an effective amount of a first agent and asecond agent, wherein the first agent is as defined in Formula I, IA,II, IIA, III, or IIIA and the second agent is for the prevention ortreatment of any one of disorder or disease indicated hereinbefore. Asused herein, the term “concomitant” or “concomitantly” as in the phrases“concomitant therapeutic treatment” or “concomitantly with” includesadministering a first agent in the present of a second agent. Aconcomitant therapeutic treatment method includes methods in which thefirst, second, third or additional agents are co-administered. Aconcomitant therapeutic treatment method also includes methods in whichthe first or additional agents are administered in the presence of asecond or additional agents, wherein the second or additional agents,for example, may have been previously administered. A concomitanttherapeutic treatment method may be executed step-wise by differentactors. For example, one actor may administer to a subject a first agentand as a second actor may administer to the subject a second agent andthe administering steps may be executed at the same time, or nearly thesame time, or at distant times, so long as the first agent (and/oradditional agents) are after administration in the presence of thesecond agent (and/or additional agents). The actor and the subject maybe the same entity (e.g. a human).

Accordingly, the invention also relates to a method for preventing,reducing or eliminating a symptom or complication of any one of theabove mentioned disease or condition. The method comprisesadministering, to a subject in need thereof, a first pharmaceuticalcomposition comprising at least one compound of the invention and asecond pharmaceutical composition comprising one or more additionalactive ingredients, wherein all active ingredients are administered inan amount sufficient to inhibit, reduce, or eliminate one or moresymptoms or complications of the disease or condition to be treated. Inone aspect, the administration of the first and second pharmaceuticalcomposition is temporally spaced apart by at least about two minutes.Preferably the first agent is a compound of Formula I, IA, II, IIA, III,or IIIA as defined herein, or a pharmaceutically acceptable saltthereof. The second agent may be selected from the following list ofcompounds: Z-Asp-Phg-Val-Aspmethyl vinyl sulfone (53)

F) Screening Assays

The compounds of the present invention may also be used in screeningmethods. For instance, these compounds may be used in methods fortracking activity of caspases in vitro and/or in vivo. The compounds ofthe present invention may also be helpful for identifying othercompounds that bind to a caspase active side. In some embodiments, thecompounds of the invention are labeled or tagged (e.g. fluorescently orradioactively labeled, affinity tag). Fluorescent or radiolabeledcompounds may also be useful in diagnostic assays.

There are a number of ways in which to determine the binding of acompound of the present invention to the caspase. In one embodiment thecaspase is bound to a support, and a labeled compound of the inventionis added to the assay. Alternatively, the compound of the invention maybe bound to the support and the caspase is added.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates or test compounds. As used herein, theterms “drug candidate” or “test compounds” are used interchangeably anddescribe any molecule, for example, protein, oligopeptide, small organicmolecule, polysaccharide, polynucleotide, and the like, to be tested forbioactivity.

Typically, the signals that are detected in the assay (e.g. in vitro, invivo and/or diagnostic) may include fluorescence, resonance energytransfer, time resolved fluorescence, radioactivity, fluorescencepolarization, plasma resonance, or chemiluminescence and the like,depending on the nature of the label. Detectable labels useful inperforming screening assays in this invention include a fluorescentlabel such as Fluorescein, Oregon green, dansyl, rhodamine, tetramethylrhodamine, texas red, Eu³⁺; a chemiluminescent label such as luciferase;colorimetric labels; enzymatic markers; or radioisotopes such astritium, I¹²⁵ and the like. Affinity tags, which may be useful inperforming the screening assays of the present invention include bebiotin, polyhistidine and the like.

F) Kits

The compound(s) of the invention may be packaged as part of a kit,optionally including a container (e.g. packaging, a box, a vial, etc).The kit may be commercially used according to the methods describedherein and may include instructions for use in a method of theinvention. Additional kit components may include acids, bases, bufferingagents, inorganic salts, solvents, antioxidants, preservatives, or metalchelators. The additional kit components are present as purecompositions, or as aqueous or organic solutions that incorporate one ormore additional kit components. Any or all of the kit componentsoptionally further comprise buffers.

The compound(s) of the invention may or may not be administered to apatient at the same time or by the same route of administration.Therefore, the methods of the invention encompass kits which, when usedby the medical practitioner, can simplify the administration ofappropriate amounts of two or more active ingredients to a patient.

A typical kit of the invention comprises a unit dosage form of a atleast one compound according to the invention, e.g., a compound ofFormula I, IA, II, IIA, III, or IIIA as defined herein or apharmaceutically acceptable salt thereof, and a unit dosage form of atleast one additional active ingredient. Examples of additional activeingredients that may be used in conjunction with the compounds accordingto the invention, include, but are not limited to any of the compoundsthat could be used in combination with the compound(s) of the inventionas indicated herein before.

Kits of the invention can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, inhalers, enemas, anddispensers for the administration of suppository formulations.

Kits of the invention can further comprise pharmaceutically acceptablevehicles that can be used to administer one or more active ingredients.For example, if an active ingredient is provided in a solid form thatmust be reconstituted for parenteral administration, the kit cancomprise a sealed container of a suitable vehicle in which the activeingredient can be dissolved to form a particulate-free sterile solutionthat is suitable for parenteral administration. Examples ofpharmaceutically acceptable vehicles are provided hereinbefore.

Headings are included herein for reference and to aid in locatingcertain sections These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specificationThus, the present invention is not intended to be limited to theembodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, concentrations, properties, and soforth used in the specification and claims are to be understood as beingmodified in all instances by the term “about.” At the very least, eachnumerical parameter should at least be construed in light of the numberof reported significant digits and by applying ordinary roundingtechniques. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the present specification and attached claimsare approximations that may vary depending upon the properties sought tobe obtained. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the embodiments are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containcertain errors resulting from variations in experiments, testingmeasurements, statistical analyses and such.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents are considered to be within the scope of this inventionand covered by the claims appended hereto. The invention is furtherillustrated by the following examples, which should not be construed asfurther limiting.

EXAMPLES

The Examples set forth herein below provide exemplary methods for thepreparation of certain representative compounds encompassed by generalFormula I, IA, II, IIA, III, or IIIA. Some Examples provide exemplaryuses of certain representative compounds of the invention. Also providedare exemplary methods for assaying the compounds of the invention for invitro and in vivo efficacy.

Example I Synthesis of compound 4 (Cbz-Asp(O-tBu)-Ala(2′quinolyl)-ValOH)

a) Fmoc-Ala(2′-quinolyl)-Val-OAllyl

Fmoc-Ala (2′-quinolyl)-OH (0.152 g, 0.347 mmol) was solvated in DMF (1mL) and CH₂Cl₂ (0.9 mL). L-Val allylester toluene-4-sulfonate (0.115 g,1.01 eq) in 0.3 mL of CH₂Cl₂ was added, followed with 4-methylmorpholine(0.04 mL, 1.05 eq) and EDC (0.0681 g, 1.02 eq). The mixture was stirredfor 3 hours, and then it was extracted using CH₂Cl₂/brine. The organiclayer was dried over MgSO₄, filtered off and concentrated to dryness.

b) Ala(2′quinolyl)-Val-OAllyl

The Fmoc-Ala(2′-quinolyl)-Val(O-Allyl) (0.425 g, 0.737 mmol) wassolvated in CH₂Cl₂ (40 mL) followed by dropwise addition of piperidine(0.6 mL, 8.24 eq). After 40 min the mixture was evaporated to drynessunder vacuum. The product was then purified on silica using a gradientof MeOH/CH₂Cl₂ (0 to 10%) to get 0.188 g of desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 8.18 (s, 1H, NH); 8.08 (d, 1H, J=8.4 Hz);8.03 (d, 1H, J=8.47 Hz); 7.79 (d, 1H, J=7.91 Hz); 7.71-7.68 (m, 1H);7.52-7.49 (m, 1H); 7.35 (d, 1H, J=8.42 Hz); 5.92-5.85 (m, 2H); 5.34-5.22(m, 2H); 4.63-4.58 (m, 2H); 4.55-4.52 (m, 1H); 3.95-3.93 (m, 1H); 3.52(dd, 1H, J=3.95 Hz); 3.25-3.20 (m, 1H); 2.20-2.13 (m, 1H); 2.01 (m, 2H,NH₂); 0.85 (m, 6H).

c) Cbz-Asp(O-tBu)-Ala(2′-quinolyl)-Val-OAllyl

Ala(2′-quinolyl)-Val(O-Allyl) (0.054 g, 0.1519 mmol) was solvated inCH₂Cl₂ (0.7 mL) followed by addition of L-Asp (O-tBu)-OH (0.053 g, 1.08eq) and 4-methylmorpholine (0.018 mL, 1.08 eq) and finally DPC (0.025mL, 1.06 eq). The mixture was stirred for 1 hour. It was then subjectedto a liquid extraction dichloromethane/brine. The organic layer wasdried over MgSO₄, concentrated to vacuum and purified on silica using agradient of MeOH/CH₂Cl₂ (0 to 10%) to get 0.084 g of the desiredcompound.

d) Cbz-Asp(O-tBu)-Ala(2′-quinolyl)-ValOH

Cbz-Asp (OtBu)-Ala(2′-quinolyl)-Val (OAllyl) (0.082 g, 0.1241 mmol) wasdissolved in THF (3.5 mL) and rotary evaporated to dryness, the samplewas then redissolved in THF (3.5 mL) and vacuum aspirated (3*1 min ofaspiration), followed by replacement of the atmosphere with Argon.Morpholine (0.04 mL, 3.7 eq) was added, followed by Pd(PPh₃)₄ (0.0171 g,0.12 eq). The sample flask was then covered with a tin foil and keptunder stirring for 3 days under Argon. The compound was evaporated todryness and the obtained residue was subjected to purification on C₁₈using a gradient MeOH/solution of H₂O at pH=3.5 (0 to 100%) to get 0.065g of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.18 (d, 1H, J=8.4 Hz); 8.02 (d, 1H, J=8.49Hz); 7.84 (d, 1H, J=8.08 Hz); 7.63 (t, 1H, J=7.51 Hz); 7.52 (t, 1H,J=7.39 Hz); 7.41 (d, 1H, J=8.3 Hz); 7.40-7.30 (m, 5H); 5.03-4.93 (m,3H); 4.50-4.48 (m, 1H); 4.26 (d, 1H, J=4.9 Hz); 3.48-3.34 (m, 2H);2.73-2.68 (m, 1H); 2.54-2.49 (m, 1H); 2.15-2.11 (m, 1H); 1.36 (m, 9H);0.87-0.84 (m, 6H).

Example 2 Synthesis of compound 5 (Ts-Ala(2′-quinolyl)-Val-OH)

a) Ts-Ala(2′-quinolyl)-OH

To L-Ala(2′-quinolyl)-OH (0.060 g, 0.277 mmol) was added H₂O (0.4 mL)and THF (0.15 mL). The mixture was stirred for 2 min before adding TEA(0.074 ml, 1.93 eq). The mixture was allowed to reach 0° C., beforeadding Tosyl chloride (0.052 g, 1 eq) in THF (0.4 mL) in a dropwisemanner. The mixture was then allowed to warm to room temperature andstirred for 16 hours. The mixture was diluted with EtOAc (8 mL) and H₂O(2 mL), then it was acidified with HCl 1N (dropwise addition) to reachpH 3/4. The mixture was extracted with EtOAc, dried over MgSO₄ andevaporated to dryness, to get upon addition of MeOH (0.5 mL) 30 mg ofprecipitate; an other portion could be obtained from the solution.

NMR ¹H (DMSO, 400 MHz) δ: 8.30-8.23 (m, 1H, NH); 8.14 (d, 1H, J=8.37Hz); 7.89 (d, 1H, J=8.0 Hz); 7.78 (d, 1H, J=8.39 Hz); 7.70 (t, 1H, J=7.2Hz); 7.55 (t, 1H, J=7.33 Hz); 7.32-7.27 (m, 3H); 6.88 (d, 2H, J=8.05Hz); 4.28 (s, 1H); 3.4-3.03 (m, 2H); 2.13 (s, 3H).

b) Ts-Ala(2′-quinolyl)-Val-OAllyl

Ts-Ala(2′-quinolyl)-OH (0.027 g, 0.076 mmol) was solvated in CH₂Cl₂followed by addition of L-Val-OAllyl ester toluene-4-sulfonate (0.025 g,1.02 eq), 4-methylmorpholine (0.017 mL, 1.91 eq), DMAP (0.0012 g, 0.13eq) and finally EDC (0.015 g, 1.04 eq). The progress of the reaction wasfollowed by TLC. The mixture was extracted with CH₂Cl₂/brine. Theorganic layer was dried over MgSO₄, filtered off and concentrated todryness. The obtained residue was purified on silica using a gradientEtOAc/Hexane (5 to 60%) to get 10 mg of the desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 7.99 (m, 2H); 7.89 (d, 1H, J=8.93 Hz); 7.76(d, 1H, J=8.06 Hz); 7.72 (t, 1H, J=8.16 Hz); 7.64 (m, 2H); 7.53 (t, 1H,J=7.61 Hz); 7.45 (d, 1H, J=6.86 Hz); 7.16 (d, 1H, J=8.34 Hz); 7.08 (m,2H); 5.86-5.79 (m, 1H); 5.27 (dd, 1H, J=17.25 Hz, J=1.36 Hz); 5.20 (dd,1H, J=10.4 Hz, J=1.0 Hz); 4.58-4.5 (m, 2H); 4.38 (dd, 1H, J=8.78 Hz,J=5.02 Hz); 4.22 (q, 1H, J=5.58 Hz); 3.41 (dd, 1H, J=15.50 Hz, J=5.23Hz); 3.14 (dd, 1H, J=15.53 Hz, J=5.64 Hz); 2.33 (s, 3H); 2.16-2.00 (m,1H); 0.72 (d, 3H, J=6.8 Hz); 0.65 (d, 3H, J=6.85 Hz).

c) Ts-Ala(2′quinolyl)-Val-OH

The Ts-Ala(2′-quinolyl)-OAllyl (0.010 g, 0.02 mmol) was dissolved in THF(3.5 mL) and rotary evaporated to dryness, the sample was thenredissolved in THF (3.5 mL) and vacuum aspirated (3*1 min ofaspiration), followed by replacement of the atmosphere with Argon.Pd(PPh₃)₄ (0.0036 g, 0.16 eq) was added under Argon, followed byMorpholine (0.007 mL, 4.1 eq). The sample flask was then covered with atin foil and kept under stirring for 3 days under Argon. The compoundwas evaporated to dryness and the obtained residue was subjected topurification on C₁₈ using a gradient: MeOH/H₂O (15 to 100%) to get 0.006g of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.05 (d, 1H, J=8.44 Hz); 7.88-7.84 (m, 2H);7.76-7.72 (m, 1H); 7.58-7.55 (m, 1H); 7.33-7.29 (m, 3H); 6.78-6.76 (m,2H); 4.35 (dd, 1H, J=10.43 Hz, J=3.38 Hz); 4.19 (d, 1H, J=4.6 Hz);3.44-3.03 (m, 2H); 2.20-2.12 (m, 1H); 2.12 (s, 3H); 0.93-0.89 (m, 6H).

Example 3 Synthesis of compound 12(Cbz-Asp(O-tBu)-Indanylglycine-Val-OH)

a) Fmoc-Indanylglycine-Val-OAllyl

Fmoc Indanylglycine (0.54 g, 1.315 mmol) was solvated in CH₂Cl₂ (5 mL)and DMF (1.8 mL) followed with the addition of L-Val allylester (0.437g, 1 eq), 4-methylmorpholine (0.15 ml, 1.04 eq) and 4 min latter DMAP(14.5 mg, 0.09 eq) then EDC (0.265 g, 1.05 eq). The mixture was stirredfor 1 hour 45 min. Then it was extracted using EtOAc/brine. The organiclayer was dried over MgSO₄, filtered off and concentrated to dryness.

b) Indanylglycine-Val-OAllyl

Fmoc-Indanylglycine-Val-OAllyl (0.758 g, 1.31 mmol) was solvated inCH₂Cl₂ (5 mL) and DMF (1.8 mL) followed by dropwise addition ofpiperidine (1.05 mL, 8.1 eq) over 30 seconds. After 45 min, the mixturewas subjected to extraction CH₂Cl₂/brine (30/15 mL) and saturated NH₄Cl(5 mL). The organic layer was dried over MgSO₄, concentrates andpurified by silica using a gradient of MeOH/CH₂Cl₂ (0 to 5%) to get 0.45g of the desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 7.80 (d, 1H, NH); 7.20-7.17 (m, 2H);7.15-7.11 (m, 2H); 5.96-5.88 (m, 1H); 5.37-5.25 (m, 2H); 4.68-4.58 (m,3H); 3.53 (d, 1H, J=5.05 Hz); 3.12-2.8 (m, 5H); 2.27-2.20 (m, 1H); 0.98(d, 3H, J=6.85 Hz); 0.94 (d, 3H, J=6.88 Hz).

c) Cbz-Asp(O-tBu)-Indanylglycine-Val-OAllyl

Indanylglycine-Val-OAllyl (0.46 g, 1.3 mmol) and Z-L-Asp(OtBu)-OH (0.425g, 1.3 mmol) were solvated in CH₂Cl₂ (4.5 mL) followed by addition of4-methylmorpholine (1.45 mL, 1.01 eq), DMAP (14 mg, 0.09 eq) then EDC(0.251 g, 1.01 eq). The mixture was stirred for 1 hour 40 min. Then itwas extracted using CH₂Cl₂/brine. The organic layer was dried overMgSO₄, concentrates and purified by silica using a gradient ofEtOAc/Hexane (10 to 80%) to get 0.578 g of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 7.35-7.25 (m, 5H); 7.15-7.07 (m, 4H);6.0-5.92 (m, 1H); 5.36 (dd, 1H, J=17.17 Hz, J=1.429 Hz); 5.24 (dd, 1H,J=10.45 Hz, J=1.251 Hz); 5.09-5.036 (m, 2H); 4.77-4.61 (m, 2H);4.54-4.50 (m, 2H); 4.33 (d, 1H, J=6.04 Hz); 2.97-2.52 (m, 7H);2.20-2.011 (m, 1H); 1.41 (s, 9H); 0.97 (d, 3H, J=1.78 Hz); 0.96 (d, 3H,J=1.78 Hz).

d) Cbz-Asp(O-tBu)Indanylglycine-Val-OH

Z-Asp (O-tBu)-Indanylglycine-Val-(OAllyl) (0.102 g, 0.1611 mmol) wasdissolved in THF (6 mL) and rotary evaporated to dryness, the sample wasthen redissolved in THF (6 mL) and vacuum aspirated (3*1 min ofaspiration), followed by replacement of the atmosphere with Argon.Pd(PPh₃)₄ (0.017 g, 0.151 0.094 eq) was added in one shot, the flask wasthen evacuated with Argon. Morpholine (0.06 mL, 4.28 eq) was added andthe flask covered with a tin foil. The mixture was kept under stirringfor 2.5 days under Argon. The sample was evaporated to dryness and theobtained residue was subjected to purification on O₁₈ using a gradientMeOH/solution of H₂O at pH=3.5 (10 to 100%) to get 0.046 g of thedesired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 7.4-7.23 (m, 5H); 7.18-7.03 (m, 4H); 5.20-5.0(m, 2H); 4.58-4.50 (m, 2H); 4.3 (d, 1H, J=5.5 Hz); 3.0-2.7 (m, 6H);2.6-2.50 (m, 1H); 1.4 (s, 9H); 0.97 (d, 3H, J=2.06 Hz); 0.96 (d, 3H,J=2.18 Hz).

Example 4 Synthesis of compound 16 (Z-Asp(β-tert-butyl)-Phg-Val-OH)

a) Fmoc-Phg-Val-OAllyl

Fmoc-PhgOH (1 g, 2.678 mmol) was dissolved in a mix of anhydrousdichloromethane and DMF (9 ml/0.5 ml). Val(OAllyl) tosyl salt form(0.804 g, 2.44 mmol) in 1 ml of dichloromethane was added followed bydiisopropylcarbodiimide (0.414 ml, 2.44 mmol) and N-methyl morpholine(0.270 ml, 2.44 mmol). The mixture was stirred for 2.20 hrs at RT, thenfiltered off on a path of celite (1 cm) and washed with dichloromethane.The filtrate was concentrated and the obtained residue was used as acrude material for the next step.

b) Phg-Val-OAllyl

1.45 g of the previous crude material (Fmoc-Phg-Val-OAllyl) wasdissolved in a solution of 20% piperidine in dichloromethane (8.5 ml)and stirred at room temperature for 45 minutes. The mixture was thenconcentrated under vacuum, diluted with dichloromethane and filtered offthrough a path of celite (1 cm). The solvent was evaporated to drynessand then purified on silica gel, eluting first with ethyl acetate/hexane(20%) followed with a gradient of dichloromethane/methanol (0 to 10%) toget 0.72 g of the N-unprotected peptide Phg-Val-OAllyl.

c) Z-Asp(β-tert-Butyl)-Phg-Val-OAllyl

The Phg-Val-OAllyl (0.89 g, 2.74 mmol) was dissolved in anhydrousdichloromethane (7 ml). Then Z-Asp(β-tert-butyl)-OH (0.89 g, 2.74 mmol)in dichloromethane (2 ml) was added followed by diisopropyl carbodiimide(0.424 ml, 2.74 mmol). The mixture was stirred at RT for 2.45 hrs andthen diluted with dichloromethane. The organic phase was washed twicewith brine and dried over anhydrous magnesium sulphate. The solvent wasevaporated and the obtained residue was purified on silica gel(gradient: ethyl acetate/hexane) to afford 0.4 g ofZ-Asp(OtBu)-Phg-Val-OAllyl.

d) 7-Asp(β-tert-Butyl)-Phg-Val-OH

Z-Asp(β-tert-butyl)-Phg-Val-OAllyl (0.064 g; 0.107 mmol) was dissolvedunder argon in dry THF (3 ml, inhibitor free). The solvent was degassedthree times under argon before adding morpholine (28 ul, 3 eq), followedby Tetrakis (13 mg). The mixture was stirred for 3.5 days at roomtemperature. The mixture was then concentrated under vacuum (12 mbar)and purified on silica gel (gradient methanol/dichloromethane: 1 to 14%)to afford 46 mg of the desired Z-Asp(β-tert-Butyl)-Phg-Val-OH.

NMR ¹H (CD₃OD, 400 MHz) δ 7.43 (m, 2H); 7.34-7.26 (m, 8H); 5.57 (s, 1H);5.10 (s, 2H); 4.60 (dd, J=8.28; 5.57 Hz, 1H); 4.24 (s, 1H); 2.81 (m,1H); 2.58 (m, 1H); 1.41 (s, 9H); 0.96 (t, J=6 Hz, 6H).

LCMS negative (M-H)=554.2

Example 5 Synthesis of compound 20 Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-OH

a) Fmoc-Glu(O-tBu)-Val-OAllyl

Fmoc-Glu (O-tBu)-OH (0.549 g, 1.292 mmol) was solvated in CH₂Cl₂ (5 mL)followed by addition of L-Val-allyl ester toluene-4-sulfonate (0.426 g,1 eq), 4-methylmorpholine (0.145 mL, 1.02 eq), DMAP (11.53 mg, 0.13 eq)and finally EDC (0.252 g, 1.02 eq). The EDC vial was washed with CH₂Cl₂(0.5 mL*2) and added to the reaction mixture. After 1 hour 10 min ofstirring, the reaction mixture was extracted with CH₂Cl₂ (30 mL)/brine(5 mL). The organic layer was dried over MgSO₄, filtered off andconcentrated. The obtained residue was purified on silica using agradient Hex/EtOAc (0 to 40%) to get 0.396 g of the desired compound.

b) Glu(O-tBu)-Val-OAllyl

Fmoc-Glu(O-tBu)-Val(OAllyl)(0.394 g, 0.698 mmol) was solvated in CH₂Cl₂(4 mL) followed by addition of piperidine (0.550 mL, 7.98 eq). After 40min, the mixture was evaporated to dryness and co-evaporated with CH₂Cl₂(20 mL*2) followed with high vacuum for 10 min to remove the excess ofpiperidine. The sample was purified on silica with a gradient ofMeOH/CH₂Cl₂ (0 to 7%) to get 0.191 g of the desired compound.

c) Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-OAllyl

Glu-Val (OAllyl) (0.189 g, 0.55 mmol) was solvated in CH₂Cl₂ (2.5 ml)followed addition of Z-Asp (OtBu)-OH (0.187 g, 1.05 eq), DMAP (5.98 mg,0.088 eq) and 4-methylmorpholine (0.065 ml, 1.07 eq), then EDC (0.109 g,1.03 eq. The vial was rinsed with 0.5 ml of dichloromethane). Themixture was stirred for 2 hour at room temperature. Then it wasextracted using CH₂Cl₂/brine. The organic layer was dried over MgSO₄,filtered off and concentrated. The obtained residue was purified onsilica using a gradient EtOAc/Hex (10 to 100%) to get 0.300 g of thedesired compound.

d) Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-OH

Cbz Asp (O-tBu)-Glu (OtBu)-Val-(OAllyl) (0.298 g, 0.46 mmol) wasdissolved in THF (10 mL) and rotary evaporated to dryness, the samplewas then redissolved in THF (10 mL) and vacuum aspirated (3*1 min ofaspiration), followed by replacement of the atmosphere with Argon.Pd(PPh₃)₄ (0.052 g, 0.1 eq) was added in one shot, the flask was thenevacued with Argon. Morpholine (0.14 mL, 3.49 eq) was added and theflask covered with a tin foil. The mixture was kept under stirring for2.5 days under Argon. The sample was evaporated to dryness and theobtained residue was subjected to purification on C₁₈ using a gradientMeOH/solution of H₂O at pH=3.5 (10 to 100%) to get 0.150 g of thedesired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 7.4-7.25 (m, 5H); 5.20-5.05 (m, 2H); 4.6-4.05(m, 1H); 4.47-4.38 (m, 1H); 4.25-4.18 (m, 1H); 2.88-2.75 (m, 1H);2.68-2.55 (m, 1H); 2.42-2.25 (m, 2H); 2.22-2.08 (m, 2H); 1.96-1.80 (m,1H); 1.45 (2s, 18H); 0.93 (t, 6H).

Example 6 Synthesis of compound 23 (Asp(β-tert-butyl)Chlorovinyl-methylvinyl sulfone tosyl salt)

a) Diethyl chloro(methylsulfone) methylphosphonate

Diethyl (methylthio)methylphosphonate (1.45 g, 6.25 mmol) was solvatedin acetic acid (5 mL, 14 eq) follow by addition of hydrogen peroxide(1.98 mL, 2.8 eq). The sample was then placed in a oil bath preheated to70° C., vigor evolution of gas shortly followed. After 30 min thereaction was allowed to reach room temperature. Then NaHCO₃ was added ina small portion until the pH become neutral. The sample was then vacuumaspirated followed by extraction with Ether (30 mL). The organic layerwas then washed with 20% of citric acid (5 mL) and then brine (2*5 mL).The organic layer was dried over MgSO₄ and purified on silica using agradient EtOAc/Hexane (20 to 100%) to get 0.552 g of the desiredcompound.

NMR ¹H (CDCl₃, 400 MHz) δ: 4.21-4.13 (m, 4H); 2.88-2.75 (m, 1H); 2.68(d, 2H, J=12.83 Hz); 2.29 (s, 3H); 1.34 (t, 6H).

b) Boc-Asp (β-tert-butyl)αchlorovinyl methylsulfone

Diethyl chloro(methylsulfone)methylphosphonate (0.327 g, 1.24 mmol) wassolvated in THF (5 mL) and the solution was allowed to reach −78° C. NaH60% (0.0519 g, 1.04 eq), which had been washed with Ether anhydrous(3*0.9 mL), was then added in suspension in THF (1.5 mL). The vialcontaining NaH in suspension was washed with THF (0.4 mL*2) and it wasadded to the solution. The mixture was stirred for 25 min, then Boc Asp(O-tBu)-H (0.326 g, 1 eq) solvated in THF (3 mL) was added dropwise tothe solution over 1 min. The vial was rinsed with THF (0.3 mL*2) andadded to the reaction mixture. After 45 min of stirring, the solutionwas quenched with a solution of saturated ammonium chloride (5 mL) andextracted with EtOAc (30 mL). The organic layer was washed with brine,dried over MgSO₄, filtered and concentrated. The residue was purified onsilica using a gradient of EtOAc/Hex (0 to 40%) to get first the cisisomer and then 0.183 g of the trans isomer compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 7.1 (d, 1H, J=8.06 Hz); 5.5 (m, 1H); 4.8 (m,1H); 3.05 (, 3H); 2.62 (m, 2H); 1.45 (2s, 18H).

c) Asp(β-tert-butyl)αchlorovinyl methylsulfone tosyl salt

Boc-Asp (β-tert-butyl)Chlorovinyl-methyl vinyl sulfone (0.182 g, 0.4937mmol) was solvated in CH₂Cl₂ (0.7 mL) followed by the addition of Et₂O(0.7 mL). p-Toluene sulfonic Acid monohydrate (0.101 g, 1.07 eq) wasadded in one shot. After 15 hours of stirring at room temperature, itwas then diluted with Ether (8 mL) and filtered off. The while solid wasthen dried over vacuum, 0.12 g of the desired compound was obtained.

NMR ¹H (DMSO, 400 MHz) δ: 8.2 (bs, 3H, NH₃); 7.48 (d, 2H, J=8.04 Hz);7.12 (d, 2H, J=7.89 Hz); 7.02 (d, 1H, J=9.4 Hz); 4.4-4.3 (m, 1H); 3.2(s, 3H); 2.85 (dd, 1H, J=16.39 Hz, J=5.55 Hz); 2.75 (dd, 1H, J=16.47 Hz,J=7.63 Hz); 2.28 (s, 3H); 1.42 (s, 9H).

Example 7 Synthesis of compound 26 (Asp(β-tert-butyl)αchlorovinylphenylsulfone tosyl salt)

a) Diethyl chloro(phenylsulfone)methylphosphonate

Diethyl chloro(phenylthio)methylphosphonate (0.919 g, 3.12 mmol) wassolvated in acetic acid (1.8 mL), followed by addition of hydrogenperoxide (0.78 mL, 2.8 eq). The sample was then placed in a oil bathpreheated to 70° C. An additional portion of hydrogen peroxide (0.21 mL)was added after 5 min. After 30 min the reaction was allowed to reachroom temperature. The sample was extracted with AcOEt/5% NaHCO₃ (30/5mL). The organic layer was dried over MgSO₄, filtered and concentrated.The residue was purified on silica using a gradient EtOAc/Hexane (10 to80%) to get 0.627 g of the desired compound.

b) Asp(β-tert-butyl)αchlorovinyl phenylsulfone

Diethyl chloro(phenylsulfone)methylphosphonate (0.458 g, 1.03 eq) wassolvated in THF (3 mL) and the solution was allowed to reach −78° C. NaH60% (0.016 g, 1.11 eq), which had been washed with Ether anhydrous(3*0.9 mL), was then added in suspension in THF (1.5 mL). The vialcontaining NaH in suspension was washed with THF (0.4 mL*2) and added tothe solution. The mixture was stirred for 25 min, then BocAsp(OtBu)-H(0.372 g, 1.36 mmol) solvated in THF (0.5 mL) was added dropwise to thesolution over 1 min. The vial was rinsed with THF (2*0.5 ml) and addedto the reaction mixture. After 45 min of stirring, the solution wasquenched with a solution of ammonium chloride saturated (5 mL) andextracted with EtOAc (30 mL). The organic layer was washed with brine,dried over MgSO₄, filtered and concentrated. The residue was purified onsilica using a gradient of Hex/EtOAc (0 to 30%), repurified using agradient of MeOH/CH₂Cl₂(0 to 3%) to elute first the cis product and thenthe trans product. Finally, a small amount of the desired compound wasobtained.

NMR ¹H (CDCl₃, 400 MHz) δ: 8.13 (m, 2H); 7.67 (t, 1H, J=7.42 Hz); 7.57(t, 2H); 6.55 (d, 1H, J=9.19 Hz); 5.75 (bs, 1H); 5.64 (bs, 1H);2.86-2.77 (m, 2H); 1.47 (s, 9H); 1.45 (s, 9H).

c) Asp(β-tert-butyl)αchlorovinyl phenylsulfone tosyl salt

Asp(β-tert-butyl)αchlorovinyl phenylsulfone (0.018 g, 0.04 mmol) wassolvated in CH₂Cl₂ (0.1 mL) followed by the addition of Et₂O (0.1 mL).p-Toluene sulfonic Acid monohydrate (0.05 g, 0.1 eq) was added in oneshot. After 15 hours of stirring at room temperature, it was thenevaporated to dryness without heating to get 18 mg of the desiredcompound.

NMR ¹H (DMSO, 400 MHz) δ: 8.3 (s, 3H, NH₃); 8.08-8.02 (m, 2H); 7.8 (t,1H, J=7.46 Hz); 7.76 (t, 2H, J=8.08 Hz); 7.48 (d, 2H, J=8.06 Hz); 7.1(d, 2H, J=7.81 Hz); 6.73 (d, 1H, J=10.09 Hz); 5.3 (m, 1H); 2.87 (dd, 1H,J=16.67 Hz, J=6.13 Hz); 2.78 (dd, 1H, J=16.77 Hz, J=7.01 Hz); 2.28 (s,3H); 1.43 (s, 9H).

Example 8 Synthesis of compound 30 (Asp(β-Methyl)methyl vinyl sulfonetosyl salt)

a) Boc-Aspartimol(β-Methyl)

a-Boc-L-Asp (β-Methyl)-OH (2.5 g, 0.0101 mol) was solvated in ethylacetate (12.5 ml) and shilled at 0° C. N-hydroxysuccinimide (1.163 g, 1eq) was added, followed with a dropwise addition of DCC (10.1 ml, 1 eq,1M in CH₂Cl₂). The mixture was allowed to reach room temperatureovernight (20 h), it was then diluted with ethyl acetate and filteredoff on celite and washed with ethyl acetate (80 ml total volume). Theorganic layer was washed with 5% NaHCO₃ (2*15 ml), brine (2*25 ml),dried over MgSO₄ and concentrated to dryness to get 3.58 g of thedesired compound.

b-Boc-L-Asp (β-Methyl)-N-hydroxysuccinimide (1.81 g, 0.00526 mol) wasdissolved in 24 mL of anhydrous THF under Argon. The mixture was chilledto 0° C., NaBH₄ (0.5 g, 2.51 eq) was added portion wise over a period of25 min. The mixture was allowed to reach room temperature, and stirredfor an extra 4 hours. A solution of ice water/brine (1/1, 15 mL) wasadded dropwise at 0° C. followed by caution addition of citric acid (0.5M, 40 mL). The biphasic mixture was stirred and the product wasextracted with EtOAc (4*40 mL). The combined organic layer were washedwith 5% NaHCO₃ (15 mL) and brine (15 mL), dried over MgSO₄, filtered offand concentrated under vacuum. The crude material was then purified onsilica with a gradient of CH₂Cl₂/MeOH (0 to 5%) to get 0.7 g of thedesired compound.

NMR ¹H(CDCl₃, 400 MHz) δ: 5.20 (bs, 1H); 4.02-3.95 (m, 1H); 3.88-3.64(m, 2H); 3.70 (s, 3H); 2.63 (d, 2H, J=5.86 Hz); 2.0 (bs, 1H, OH); 1.44(s, 9H).

b) Boc-Asp(β-Methyl)-H

Oxalyl chloride 2M in CH₂Cl₂ (1.242 mL, 1.7 eq) dissolved in CH₂Cl₂ (2.4mL) was cooled to −65° C. A solution of DMSO (0.4 mL, 3.9 eq) in CH₂Cl₂(0.92 mL) was added dropwise over 20 min at −65° C. Boc Asp (OCH₃)—CH₂OHin CH₂Cl₂ (2.6 mL) was added dropwise over a period of 20 min and thereaction was stirred for an extra 15 min at −65° C. TEA (1.33 mL, 6.54eq) in CH₂Cl₂ (1.42 mL) was added dropwise over 20 min. The reaction wasleft for an extra 55 min at −65° C./−70° C. then quenched at thistemperature with ether/0.5 N KHSO₄ (30/6 mL). The organic layer waswashed 3 times with 0.5N KHSO₄ (3*6 mL) then brine, dried over MgSO₄,filtered off and concentrate, to get 0.29 g of the desired compound.

NMR ¹H(CDCl₃, 400 MHz) δ: 9.65 (s, 1H); 5.61 (d, 1H, J=7.04 Hz);4.38-4.35 (q, 1H, J=4.3 Hz); 3.70 (s, 3H); 3.01 (dd, 1H, J=17.41 Hz,J=4.49 Hz); 2.83 (dd, 1H, J=17.41 Hz, J=4.69 Hz); 1.46 (s, 9H).

c) Boc-Asp(β-Methyl)methyl vinyl sulfone

NaH 95% (0.038 g, 1.2 eq), which had been washed with Ether anhydrous(3*0.9 mL), was suspended in THF (1 mL). The NaH solution was addeddropwise at 0° C. to Diethyl (methylsulfone) methylphosphonate (0.280 g,1.08 eq) which was dissolved in a solution of THF (10 mL). The mixturewas stirred for 20 min, then Boc Asp (OMethyl)-H (0.290 g, 1.255 mmol)solvated in THF (2.5 mL) was added dropwise to the solution over 1 min.After 15 min at 0° C., the reaction was allowed to reach roomtemperature. After 1 hour of stirring, the solution was quenched with asolution of saturated ammonium chloride (10 mL) and extracted with EtOAc(30 mL). The organic layer was washed with brine (20 mL), dried overMgSO₄, filtered and concentrate. The residue was purified on silicausing a gradient of EtOAc/Hex (10 to 80%) to elute first the cis isomerthen the trans isomer (0.174 g).

d) Boc-Asp(β-Methyl)methyl vinyl sulfone tosyl salt

Boc-Asp(β-Methyl)methyl vinyl sulfone (0.172 g, 0.56 mmol) was solvatedin CH₂Cl₂ (0.44 mL) followed by the addition of Et₂O (0.44 mL).p-Toluene sulfonic Acid monohydrate (0.19 g, 1.04 eq) was added in oneshot. After 15 hours of stirring at room temperature, it was thendiluted with Ether (2 mL) and filtered off. The while solid was thendried over vacuum, 0.140 g of the desired compound was obtained.

NMR ¹H (DMSO, 400 MHz) δ: 8.20 (bs, 3H, NH₃); 7.47-7.45 (m, 2H); 7.11(dd, 2H, J=8.41 Hz, J=0.58 Hz); 7.03 (dd, 1H, J=15.45 Hz, J=1.17 Hz);6.73 (dd, 1H, J=15.45 Hz, J=6.26 Hz); 4.34 (q, 1H, J=6.45 Hz); 3.66 (s,3H); 3.05 (s, 3H); 2.92-2.82 (m, 2H); 2.28 (s, 3H).

Example 9 Synthesis of compound 33 (Asp(β-tert-butyl)methyl vinylsulfone tosyl salt)

a) Diethyl (methylsulfone)methylphosphonate

Diethyl (methylthio)methylphosphonate (4.0180 g, 20.3 mmol) was solvatedin acetic acid (14 mL, 12 eq) follow by dropwise addition (10 min) ofhydrogen peroxide (5.9 mL, 2.56 eq). The sample was then heated to 70°C., vigor evolution of gas shortly followed. After 25 min the reactionwas allowed to reach room temperature. Then NaHCO₃ was added in a smallportion until the pH become neutral. The sample was then vacuumaspirated followed by extraction with Ether. The organic layer was thenwashed with 20% of citric acid (5 mL) and then brine (2*10 mL). Theorganic layer was dried over MgSO₄ and purified on silica using agradient EtOAc/Hexane (10 to 100%) to get 3.237 g of the desiredcompound.

NMR ¹H (DMSO, 400 MHz) δ: 4.18 (d, 2H, J=16.62 Hz); 4.10-4.03 (m, 4H);3.10 (s, 3H); 1.23 (t, 6H, J=7.04 Hz).

b) Boc-Asp(β-tert-butyl)methyl vinyl sulfone

NaH 60% (0.087 g, 1.11 eq), which had been washed with Ether anhydrous(3*0.9 mL), was suspended in THF (1 mL). The NaH solution was addeddropwise to Diethyl (methylsulfone)methylphosphonate (0.485 g, 1.08 eq)which was dissolved in a 0° C. solution of THF (20 mL). The mixture wasstirred for 20 min, then Boc Asp (OtBu)-H (0.537 g, 1 eq) solvated inTHF (2 mL) was added dropwise to the solution over 1 min. After 10 minat 0° C., the reaction was allowed to reach room temperature. After 1hour of stirring, the solution was quenched with a solution of ammoniumchloride saturated (45 mL) and extracted with EtOAc (100 mL). Theorganic layer was washed with brine (20 mL), dried over MgSO₄, filteredand concentrate. The residue was purified on silica using a gradient ofHex/EtOAc (10 to 80%) to elute 0.05 g of the cis compound and 0.480 g ofthe trans compound.

NMR ¹H(CDCl₃, 400 MHz) δ: 6.86 (dd, 1H, J=15.08 Hz, J=4.60 Hz); 6.51(dd, 1H, J=15.14 Hz, J=1.38 Hz); 5.41 (bs, 1H); 4.7 (bs, 1H); 2.91 (s,3H); 2.64-2.52 (qd, 2H); 1.43 (s, 18H).

c) Asp(β-tert-butyl)methyl vinyl sulfone tosyl salt

Boc-Asp(β-tert-butyl)methyl vinyl sulfone (0.158 g, 0.4534 mmol) wassolvated in CH₂Cl₂ (0.7 mL) followed by the addition of Et₂O (0.7 mL).p-Toluene sulfonic Acid monohydrate (0.0878 g, 1.02 eq) was added in oneshot. The use of excess of PTSA hydrate cleaves both boc and tert-butylgroups unlike what was reported by Palmer. After 15 hours of stirring atroom temperature, it was then diluted with Ether (5 mL) and filteredoff. The while solid was then dried over vacuum, 0.121 g of the desiredcompound was obtained.

NMR ¹H (DMSO, 400 MHz) δ: 8.18 (bs, 3H, NH₃); 7.47 (d, 2H, J=8.14 Hz);7.10 (d, 2H, J=7.85 Hz); 7.02 (d, 1H, J=15.42 Hz); 6.70 (dd, 1H, J=15.40Hz, J=6.61 Hz); 4.27 (q, 1H, J=6.3 Hz); 3.04 (s, 3H); 2.81-2.70 (qd,2H); 2.28 (s, 3H); 1.42 (s, 9H).

Example 10 Synthesis of compound 37 (Asp-vinyl phenyl sulfone salt form)

The commercially available N-tBoc-L-Asp(β-tert-Butyl)-O-succinimide wasreduced to the corresponding alcohol in the presence of sodiumborohydride in THF, as described in the literature (Ramond J. Begeron etal., 1999)

a) Boc-Asp(β-tert-Butyl)-H

The alcohol is then oxidized to the correspondingBoc-L-Asp(β-tert-Butyl)-H in the presence of oxalyl chloride, DMSO andTEA in dichloromethane at −70° C. as described in the literature(William R. Ewing et al., 1999; Won Bum Jang. 2004 and Mancuso A et al.,1981)

b) Diethyl phenylsulfonylmethylphosphonate

The precursor of phenyl vinyl sulfone was obtained in one step frombenzenesulfonyl fluoride and triethyl phosphorane in the presence oflithium hexamethyldisilazide at −78° C. to get diethylphenylsulfonylmethylphosphonate as described in the literature (Won BumJang et al., 1998).

Boc-Asp(β-tert-Butyl)-vinyl phenyl sulfone (Gang Wang et al., 2003;Marion G. Gotz et al., 2004; Palmer, James T et al., 1995).

c) Boc-Asp-vinyl phenyl sulfone

Sodium hydride (40 mg (60%), 1.09 eq) was added to a solution of diethylphenylsulfonylmethylphosphonate (0.279 g, 1.09 eq) in dry THF (5.6 ml)at 0° C. The mixture was stirred for 20 minutes before adding,drop-wise, a solution of Boc-L-Asp(O-t-Bu)-H (0.24 g, 0.876 mmol) in 1.6ml of THF. The mixture was stirred for 1.15 h at RT, then poured into amix of ethyl acetate and ammonium chloride saturated solution (45/15ml). The organic layer was dried over magnesium sulfate and the solventwas evaporated to dryness. The crude material was purified on silica gel(gradient: ethyl acetate/hexane) to make the desired compound with ahigh chemical yield.

NMR ¹H (CD₃OD, 400 MHz) δ 7.87 (d, J=7.62 Hz, 2H); 7.61 (t, J=7.35 Hz,1H); 7.53 (t, J=7.68 Hz, 2H); 6.92 (dd, J=15.08 and 4.28 Hz, 1H); 6.46(d, J=15.12 Hz, 1H); 5.34 (m, 1H); 4.68 (m, 1H); 2.63-2.52 (m, 2H); 1.40(s, 18H).

d) Asp Vinyl phenyl sulfone tosyl salt

Boc-L-Asp(β-tert-Butyl)-Vinyl phenyl sulfone (0.1 g, 0.243 mmol) wasdissolved in a mix of dichloromethane and ether (0.7/0.7 ml), then PTSAhydrate (1 eq) was added. The use of excess of PTSA hydrate cleaves bothboc and tert-butyl groups unlike what was reported by Palmer. Themixture was stirred at room temperature overnight. Then, it was dilutedwith ether (8 ml). The white precipitate was filtered off and dried toyield to the desired compound as a white powder.

Example 11 Synthesis of compound 40 (Asp(β-tert-butyl)phenoxyvinylsulfone tosyl salt)

To a solution of phenol (1.62 g, 17.2 mmol) in Et₂O (50 mL) at −10° C.was added TEA (3.6 mL, 1.5 eq). After 15 min a solution ofmethanesulfonylchloride (1.6 mL, 1.2 eq) in Et₂O (4 mL) was addeddropwise over 50 min. Then the solution was allowed to warm at roomtemperature and an additional portion of Et₂O (5 mL) was added. Thereaction was quenched by addition of 1N HCl (12 mL, 4 C), the organiclayer was then washed with saturated NaHCO₃, brine and dried over MgSO₄,filtered off and concentrate to give an oil which was recrystallised forCH₂Cl₂/Hex (1/1). The resulting solid was filtered and the residualsolvent removed.

a) Diethyl (phenoxysulfone)methylphosphonate

To a solution of methanesulfonyl phenoxy (1.938 g, 11.25 mmol) in THF (8mL) at −78° C., was added dropwise a solution of potassiumbis(trimethylsilyl)amide (2.36 g, 1 eq) in 11 mL of THF over a period of40 min. Then the reaction was stirred for an extra 5 min. Diethylchlorophosphonate (0.95 mL, 0.59 eq) was added dropwise over 7 min.After 1 hour, the reaction was quenched by dropwise addition of asolution of Acetic Acid (0.645 mL, 0.59 eq) over 5 min. The solution wasallowed to warm to room temperature and the solvent was removed invacuum. The product was extract with CH₂Cl₂ (30 mL) and H₂O (10 mL),dried over MgSO₄, filtered off and concentrate. The residue was purifiedon silica using a gradient EtOAc/Hexane (12 to 100%) to get 1.046 g ofthe desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 7.44-7.41 (m, 2H); 7.36-7.32 (m, 3H);4.31-4.25 (m, 4H); 3.81 (d, 2H, J=17.14 Hz); 1.38 (t, 6H, J=7.11 Hz).

b) Asp(β-tert-butyl)phenoxy vinyl sulfone

Diethyl (phenoxysulfone)methylphosphonate (0.36 g, 1.07 mmol) wassolvated in THF (10 mL) and the solution was allowed to reach −0° C. NaH60% (0.0539 g, 1.22 eq), which had been washed with Ether anhydrous(3*0.9 mL), was then added in suspension in THF (1 mL). The vialcontaining NaH in suspension was washed with THF (1 mL) and it was addedto the solution. The mixture was stirred for 20 min, then Boc Asp(O-tBu)-H (0.30 g, 1 eg) solvated in THF (2 mL) was added dropwise tothe solution over 1 min. The vial was rinsed with THF (1 mL) and addedto the reaction mixture. After 2 hours of stirring at room temperature,the solution was quenched with a solution of ammonium chloride saturated(5 mL) and extracted with EtOAc (30 mL). The organic layer was washedwith brine, dried over MgSO₄, filtered off and concentrated. The residuewas purified on silica using a gradient of EtOAc/Hexane (0 to 30%, 30 to80%) to get 0.359 g of the desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 7.38 (t, 2H, J=7.96 Hz); 7.29 (t, 1H, J=7.46Hz); 7.23-7.21 (m, 2H); 6.79 (dd, 1H, J=15.14 Hz, J=4.67 Hz); 6.49 (dd,1H, J=15.18 Hz, J=1.31 Hz); 5.37 (bs, 1H); 4.65 (bs, 1H); 2.60 (dd, 1H,J=16.04 Hz, J=5.46 Hz); 2.50 (dd, 1H, J=16.04 Hz, J=5.63 Hz); 1.46 (s,9H); 1.43 (s, 9H).

c) Asp(β-tert-butyl)phenoxy vinyl sulfone tosyl salt

Asp(β-tert-butyl)phenoxy vinyl sulfone (0.187 g, 0.444 mmol) wassolvated in CH₂Cl₂ (0.7 mL) followed by the addition of Et₂O (0.7 mL).p-Toluene sulfonic Acid monohydrate (0.084 g, 1.01 eq) was added in oneshot. After 15 hours of stirring at room temperature, it was thendiluted with Ether (8 mL) and filtered off. The while solid was thendried over vacuum to get the desired compound.

NMR ¹H (DMSO, 400 MHz) δ: 8.25 (bs, 3H, NH₃); 7.49-7.46 (m, 4H); 7.39(t, 1H, J=7.4 Hz); 7.31-7.29 (m, 2H); 7.22-7.17 (m, 1H); 7.10 (d, 2H,J=7.88 Hz); 6.82 (dd, 1H, J=15.41 Hz, J=6.15 Hz); 4.35 (m, 1H);2.82-2.73 (m, 2H); 2.28 (s, 3H); 1.41 (s, 9H).

Example 12 Synthesis of compound 43 (Asp(β-tert-butyl)isopropyl vinylsulfone tosyl salt)

Chloromethyl isopropylsulfide (12.54 g, 100.6 mmol) was heated to 110°C. follow by dropwise addition of triethylphosphonyl (21 mL, 1.10 eq).After stirring for 8 hours, the reaction was allowed to reach to roomtemperature. The sample was purified by distillation (110° C./6 mmbar)to get 4.7 g of Diethyl (isopropylthio)methylphosphonate.

NMR ¹H (CDCl₃, 400 MHz) or 4.21-4.13 (m, 4H); 3.19-3.14 (m, 1H); 2.76(d, 2H, J=14.35 Hz); 1.34-1.27 (m, 12H).

Example 13 Synthesis of compound 41(Diethyl(isopropylsulfone)methylphosphonate)

Diethyl (isopropylthio)methylphosphonate (4.746 g, 0.02 mol) wassolvated in acetic acid (14.5 mL, 12.06 eq) followed by dropwiseaddition over 5 min of hydrogen peroxide (6 mL, 2.52 eq). The sample wasthen heated to 70° C., vigor evolution of gas shortly followed. After 30min the reaction was allowed to reach room temperature. Then NaHCO₃ wasadded in a small portion until the pH become neutral. The sample wasthen vacuum aspirated followed by extraction with Ether (30 mL). Theorganic layer was then washed with 20% of citric acid (5 mL) and thenbrine (2*10 mL). The organic layer was combined and extract withCH₂Cl₂/water, dried over MgSO₄ and purified on silica using a gradientEtOAc/Hexane (20 to 100%) to get 4.689 g of the desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 4.23-4.50 (m, 4H); 3.67-3.63 (m, 1H); 3.53(d, 2H, J=16.82 Hz); 1.37-1.30 (m, 12H).

Example 14 Synthesis of compound 42 (BocAsp(β-tert-butyl)isopropyl vinylsulfone)

Diethyl (isopropylsulfone)methylphosphonate (0.036 g, 0.1427 mmol) wassolvated in THF (0.2 mL) and the solution was allowed to reach 0° C. NaH60% (5.38 mg, 1.05 eq), which had been washed with Ether anhydrous(3*0.9 mL), was then added in suspension in THF (1.2 mL). The mixturewas stirred for 25 min, then Boc Asp (O-tBu)-H (0.0351 g, 1 eq) solvatedin THF (0.6 mL) was added dropwise to the solution over 1 min. After 45min of stirring, the solution was quenched with a solution of ammoniumchloride saturated (2 mL) and extracted with EtOAc (10 mL). The organiclayer was washed with brine, dried over MgSO₄, filtered andconcentrated. The residue was purified on silica using a gradient ofEtOAc/Hexane (5 to 60%) to get 29.3 mg of the desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 6.82 (dd, 1H, J=15.24 Hz, J=4.78 Hz); 6.38(dd, 1H, J=15.17 Hz, J=1.41 Hz); 5.43 (m, 1H); 4.67 (s, 1H); 3.07-3.0(m, 1H); 2.64 (dd, 1H, J=16.07 Hz, J=5.5 Hz); 2.57 (dd, 1H, J=16.12 Hz,J=5.52 Hz); 1.48-1.32 (m, 24H).

Example 15 Synthesis of compound 43 (Asp(β-tert-butyl)isopropyl vinylsulfone tosyl salt)

Boc-Asp(β-tert-butyl)isopropyl vinyl sulfone (0.028 g, 0.0741 mmol) wassolvated in CH₂Cl₂ (0.2 mL) followed by the addition of Et₂O (0.2 mL).p-Toluene sulfonic Acid monohydrate (0.0154 g, 1.09 eq) was added in oneshot. After 15 hours of stirring at room temperature, it was thendiluted with Ether (8 mL) and filtered off. The while solid was thendried over vacuum, 11 mg of the desired compound was obtained.

NMR ¹H (DMSO, 400 MHz) δ: 8.21 (bs, 3H, NH₃); 7.46 (d, 2H, J=7.81 Hz);7.10 (d, 2H, J=7.85 Hz); 6.92-6.85 (m, 1H); 6.71 (dd, 1H, J=15.42 Hz,J=6.34 Hz); 4.30 (m, 1H); 3.23-3.13 (m, 1H); 2.85-2.72 (m, 2H); 2.28 (s,3H); 1.42 (s, 9H); 1.21 (m, 6H).

Example 16 Synthesis of compound 46 (Asp(β-tert-butyl)morpholine vinylsulfone tosyl salt)

To a solution of morpholine (1.4 mL, 16.07 mmol) in CH₂Cl₂ (30 mL) at−10° C. was added TEA (3.4 mL, 1.5 eq). After 15 min a solution ofmethanesulfonylchloride (1.5 mL, 1.2 eq) in CH₂Cl₂ (4 mL) was addeddropwise over 40 min. Then the solution was allowed to warm at roomtemperature and an additional portion of Et₂O (5 mL) was added. Thereaction was quenched by addition of 1N HCl (12 mL, 4 C), the organiclayer was then washed with saturated NaHCO₃, brine and dried over MgSO4,filtered off and concentrate to give an oil which was purified on silicausing a gradient of MeOH/CH₂Cl₂ (0 to 5%) to get 0.831 g of Diethyl(morpholinethio)methylphosphonate.

Example 16 Synthesis of compound 44 (Diethyl(morpholinesulfone)methylphosphonate)

To a solution of methane sulfonyl morpholine (0.719 g, 4.356 mmol) inTHF (4 mL) at −78° C., was added dropwise a solution of potassiumbis(trimethylsilyl)amide (0.748 g, 0.86 eq) in 11 mL of THF over aperiod of 40 min. Then the reaction was stirred for an extra 5 min.Diethyl chloromethylphosphonate (0.37 mL, 0.57 eq) was added dropwiseover 7 min. After 1 hour, the reaction was quenched by dropwise additionof a solution of Acetic Acid (0.226 mL, 0.77 eq) over 5 min. Thesolution was allowed to warm to room temperature and the solvent wasremoved in vacuum. The product was extract with CH₂Cl₂ (30 mL) and H₂O(10 mL), dried over MgSO₄, filtered off and concentrate. The residue waspurified on silica using a gradient MeOH/CH₂Cl₂ (0 to 4%) to get 0.247 gof the desired compound.

NMR ¹H (CDCl₃, 400 MHz) δ: 4.26-4.20 (m, 4H); 3.76 (t, 4H, J=4.6 Hz);3.51 (d, 2H, J=17.33 Hz); 3.34 (t, 4H, J=4.74 Hz); 1.37 (t, 6H, J=7.11Hz).

Example 17 Synthesis of compound 45 (Boc-Asp(β-tert-butyl)morpholinevinyl sulfone)

Diethyl (morpholinesulfone)methylphosphonate (0.103 g, 1.07 mmol) wassolvated in THF (1 mL) and the solution was allowed to reach −10° C. NaH60% (0.013 g, 1.04 eq), which had been washed with Ether anhydrous(3*0.9 mL), was then added in suspension in THF (1 mL). The vialcontaining NaH in suspension was washed with THF (0.5 mL) and it wasadded to the solution. The mixture was stirred for 20 min, then Boc Asp(OtBu)-H (0.208 g, 1 eq) solvated in THF (1.5 mL) was added dropwise tothe solution over 1 min, the vial was washed with DMF (0.5 ml) and addedto the solution. After 3 hours of stirring at room temperature, thesolution was quenched with a solution of ammonium chloride saturated (5mL) and extracted with EtOAc (30 mL). The organic layer was washed withbrine, dried over MgSO4, filtered off and concentrated. The residue waspurified on silica using a gradient of first Hex/EtOAc (5 to 80%) thenCH₂Cl₂/MeOH (0 to 10%) to get 0.075 g of the desired compound.

Example 18 Synthesis of compound 46 (Asp(β-tert-butyl)morpholine vinylsulfone tosyl salt)

Boc-Asp(β-tert-butyl)morpholine vinyl sulfone (0.075 g, 0.178 mmol) wassolvated in CH₂Cl₂ (0.2 mL) followed by the addition of Et₂O (0.2 mL).p-Toluene sulfonic Acid monohydrate (0.034 g, 1.0 eq) was added in oneshot. After 15 hours of stirring at room temperature, it was thendiluted with Ether (0.5 mL) and filtered off. The while solid was thendried over vacuum, 0.045 g of the desired compound was obtained.

NMR ¹H (DMSO, 400 MHz) δ: 8.20 (bs, 3H, NH₃); 7.47 (d, 2H, J=7.9 Hz);7.10 (d, 2H, J=7.89 Hz); 6.88 (d, 1H, J=15.33 Hz); 6.60 (dd, 1H, J=15.00Hz, J=6.36 Hz); 4.28 (s, 1H); 3.65 (t, 4H, J=4.43 Hz); 3.02 (m, 4H);2.84-2.76 (m, 2H); 2.28 (s, 3H); 1.42 (s, 9H).

Example 18 Synthesis of compound 47(Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)αchlorovinylmethylsulfone)

The Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-OH (18 mg, 0.029 mmol) isdissolved in a mix of dichloromethane and DMF (0.39 ml/0.13 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (8 μl) followed 3 min latter with isobutyl chloroformate (7μl). The mixture was stirred at −15° C. for 10 minutes. Next, Asp(β-tert-butyl)αchloromethyl-methylsulfone tosyl salt (13 mg, 1 eq) wasadded in one shot, followed by N-methyl morpholine (8 μl). The mixturewas stirred 35 minutes at −15/−20 and then diluted with 5 ml ofdichloromethane and quenched with 1.5 ml of a saturated solution ofsodium bicarbonate. The organic layer was dried over magnesium sulphate,the solvent was evaporated to dryness. The obtained residue was purifiedon silica gel (gradient: ethyl acetate/hexane: 5 to 100%) to afford 12mg of the desired compound.

Example 19 Synthesis of compound 48(Z-Asp-Ala(2′-quinolyl)-Val-Asp-αchlorovinyl methylsulfone)

Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)-αchlorovinylmethylsulfone (11.7 mg) was dissolved in dichloromethane (0.24 ml),followed by addition of trifluoroacetic acid (0.35 ml). The mixture wasstirred at room temperature overnight (15 h). It was then quenched withdiethyl ether (5 ml), the solvent was removed under vacuum. The obtainedresidue was diluted again with ether (5 ml) and the process was repeatedtwice. The precipitate was washed with 2*1 ml of ether, dried to give 9mg of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.6-7.25 (m, 11H); 7.07 (d, 1H, J=8.61 Hz);5.03-4.06 (m, 4H); 4.45-4.42 (m, 1H); 4.09-4.05 (m, 1H); 3.65-3.49 (m,2H); 3.07 (s, 3H); 2.84-2.7 (m, 2H); 2.71-2.66 (m, 2H); 2.00 (m, 1H);0.85 (t, 6H, J=8.2 Hz).

LCMS (M-H+)=772.4.

Example 20 Synthesis of compound 49(Ts-Ala(2′quinolyl)-Val-Asp(β-tert-Butyl)-αchlorovinyl methylsulfone)

The Ts-Ala (2′-quinolyl)-OH (5.6 mg, 0.0123 mmol) was dissolved in a mixof CH₂Cl₂/DMF (0.15/0.13 mL). The mixture was allowed to reach −15°C./−20° C. (ice MeOH bath) before adding NMM (0.004 mL) followed 3 minlater with isobutylchloroformate (0.004 mL). The mixture was stirred for10 min at −15° C. before adding Asp (β-tert-butyl) Chlorovinyl-methylvinyl sulfone tosyl salt (6.3 mg, 1 eq) in one shot, followed with4-methylmorpholine (0.004 mL). The mixture was stirred at −15° C./−20°C. for 30 min, it was then diluted with CH₂Cl₂ (5 mL), then water (2 mL)and the mixture was allowed to reach room temperature, extracted. Theorganic layer was dried over MgSO₄, concentrated and purified by silicausing Hex/EtOAc (15 to 100%) to get 6 mg of the desired compound.

Example 21 Synthesis of compound 50(Ts-Ala(2′quinolyl)-Val-Asp-αchlorovinyl methylsulfone)

Ts-Ala(2′quinolyl)-Val-Asp(β-tert-Butyl)-αchlorovinyl methylsulfone (6mg) was dissolved in dichloromethane (0.15 ml), followed by addition oftrifluoroacetic acid (0.2 ml). The mixture was stirred at roomtemperature overnight (15 h). It was then quenched with diethyl ether (5ml) and the solvent was removed under vacuum. The obtained residue wasdiluted again with ether and the process was repeated twice. Theprecipitate was washed with 2*1 ml of ether, dried to give 4 mg of thedesired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.37 (m, 1H); 8.01-7.99 (m, 1H); 7.96-7.94(m, 1H); 7.90-7.94 (m, 1H); 7.90-7.88 (m, 1H); 7.71-7.63 (m, 1H); 7.46(m, 1H); 7.37 (d, 2H, J=8.22 Hz); 7.08 (d, 1H, J=8.46 Hz); 6.88 (d, 2H,J=8.0 Hz); 5.04-5.01 (m, 1H); 4.37-4.34 (m, 1H); 4.08-4.07 (m, 1H);3.44-3.4 (m, 1H); 3.2 (m, 1H); 3.08 (s, 3H); 2.83-2.78 (m, 1H);2.73-2.684 (m, 1H); 2.17 (s, 3H); 2.07-2.03 (m, 1H); 0.91-0.89 (m, 6H).

Example 22 Synthesis of compound 51(Z-Asp(β-methyl)-Indanylglycine-Val-Asp(β-methyl)methyl vinyl sulfone)

The Z-Asp(β-methyl)-Indanylglycine-Val-OH (16.7 mg, 0.0301 mmol) isdissolved in a mix of THF and DMF (0.5 ml/0.1 ml). The mixture wasallowed to reach −15/−20° C. before adding N-methyl morpholine (9 μl)followed 3 min latter with isobutyl chloroformate (8 μl). The mixturewas stirred at −15° C. for 10 minutes. Next, Asp (β-methyl)methyl vinylsulfone tosyl salt (12 mg, 1 eq) was added in one shot, the vial waswashed with THF (0.1 ml) and added to the solution, followed by theaddition of N-methyl morpholine (9 μl). The mixture was stirred 35minutes at −15/−20° C. and then diluted with 7 ml of dichloromethane andquenched with 1.5 ml of a saturated solution of sodium bicarbonate.After that, the organic layer was dried over magnesium sulphate. Thesolvent was evaporated to dryness. The obtained residue was purified onsilica gel (ethyl acetate/hexane: 40% then dichloromethane/methanol: 5to 15%) to afford 14 mg ofZ-Asp(β-methyl)-Indanylglycine-Val-Asp(β-methyl)-methyl vinyl sulfone.

NMR ¹H (DMSO, 400 MHz) δ: 8.31 (d, NH, J=7.82 Hz); 8.11 (d, NH, J=8.8Hz); 7.93 (d, NH, J=8.21 Hz); 7.69 (d, NH, J=8.02 Hz); 7.37-7.08 (m,9H); 6.74 (dd, 1H, J=15.45 Hz, J=4.30 Hz); 6.67 (d, 1H, J=15.59 Hz);5.05 (s, 2H); 4.99 (m, 1H); 4.44 (m, 2H); 4.11 (m, 1H); 3.57 (s, 6H);2.98 (s, 3H); 2.95-2.5 (m, 9H); 2.00-1.94 (m, 1H); 0.84 (t, 6H, J=6.06Hz).

Example 23 Synthesis of compound 52(Z-Asp(β-tert-Butyl)-Phg-Val-Asp(β-tert-Butyl)methyl vinyl sulfone)

The Z-Asp(β-tert-Butyl)-Phg-Val-OH (17 mg, 0.0306 mmol) is dissolved ina mix of dichloromethane and DMF (0.39 ml/0.13 ml). The mixture wasallowed to reach −15/−20° C. before adding N-methyl morpholine (8 μl)followed 3 min latter with isobutyl chloroformate (8 μl). The mixturewas stirred at −15° C. for 10 minutes. Next, Asp (p-tert-butyl)methylvinyl sulfone tosyl salt (12 mg, 1 eq) was added in one shot, followedby N-methyl morpholine (8 μl). The mixture was stirred 35 minutes at−15/−20° C. and then diluted with 5 ml of dichloromethane and quenchedwith 1.5 ml of a saturated solution of sodium bicarbonate. The organiclayer was dried over magnesium sulphate. The solvent was evaporated todryness. The obtained residue was purified on silica gel (gradient:ethyl acetate/hexane: 5 to 100%) to afford 8 mg ofZ-Asp(β-tert-Butyl)-Phg-Val-Asp(β-tert-butyl)-methyl vinyl sulfone.

Example 24 Synthesis of compound 53 (Z-Asp-Phg-Val-Asp-methyl vinylsulfone)

Z-Asp(β-tert-Butyl)-Phg-Val-Asp(β-tert-butyl)-methyl vinyl sulfone (6.2mg) was dissolved in dichloromethane (0.16 ml), followed by addition oftrifluoroacetic acid (0.22 ml). The mixture was stirred at roomtemperature overnight (15 h). It was then quenched with diethyl ether (5ml), then the solvent was removed under vacuum. The obtained residue wasdiluted again with ether (5 ml) and the process was repeated twice. Thecrude material was diluted once more with ether, the filtrate wasremoved and the precipitate was washed with 2*1 ml of ether, dried togive 5 mg of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 7.46-7.29 (m, 10H); 6.83 (dd, 1H, J=15.22 Hz,J=4.60 Hz); 6.68 (dd, 1H, J=15.229 Hz, J=1.36 Hz); 5.38 (s, 1H); 5.10(q, 2H, J=12.73 Hz); 4.94-4.90 (m, 1H); 4.56 (t, 1H, J=6.75 Hz); 4.10(d, 1H, J=7.07 Hz); 2.95 (s, 3H); 2.98-2.88 (m, 1H); 2.76-2.60 (m, 3H);2.21-2.01 (m, 1H); 0.98 (t, 6H, J=6.3 Hz).

LCMS (M-H⁺)=673.7

Example 26 Synthesis of compound 54(Z-Asp(β-tert-Butyl)-Al(2′-quinolyl)-Val-Asp(β-tert-Butyl)methyl vinylsulfone)

The Z-Asp(β-tert-Butyl)-Al(2′-quinolyl)-Val-OH (19 mg, 0.0306 mmol) isdissolved in a mix of dichloromethane and DMF (0.4 ml/0.14 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (9 μl) followed 3 min latter with isobutyl chloroformate (9μl). The mixture was stirred at −15° C. for 10 minutes. Next, Asp(β-tert-butyl)methyl vinyl sulfone tosyl salt (13.1 mg, 1 eq) was addedin one shot, followed by N-methyl morpholine (9 μl). The mixture wasstirred 35 minutes at −15/−20° C. and then diluted with 5 ml ofdichloromethane and quenched with 1.5 ml of a saturated solution ofsodium bicarbonate. After that, the organic layer was washed with sodiumbicarbonate and dried over magnesium sulphate. The solvent wasevaporated to dryness. The obtained residue was purified on silica gel(gradient: ethyl acetate/hexane: 5 to 100%) to afford 13.1 mg ofZ-Asp(β-tert-Butyl)-Al(2′-quinolyl)-Val-Asp(β-tert-butyl)-methyl vinylsulfone.

Example 27 Synthesis of compound 55(Z-Asp-Ala(2′-quinolyl)-Val-Aspmethyl vinyl sulfone)

Z-Asp(β-tert-Butyl)-Phg-Val-Asp(β-tert-butyl)-methyl vinyl sulfone (13.1mg) was dissolved in dichloromethane (0.32 ml), followed by addition oftrifluoroacetic acid (0.44 ml). The mixture was stirred at roomtemperature overnight (15 h). It was then quenched with diethyl ether (5ml), then the solvent was removed under vacuum. The obtained residue wasdiluted again with ether and the process was repeated twice. Theprecipitate was washed with 2*1 ml of ether, dried to give 12 mg of thedesired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.68 (m, 1H); 8.17 (d, 1H, J=8.9 Hz); 8.1 (d,1H, J=8.2 Hz); 7.93 (t, 1H, J=7.85 Hz); 7.78 (m, 2H); 7.34-7.29 (m, 5H);6.88 (dd, 1H, J=15.20 and 4.75 Hz); 6.7 (d, 1H, J=15.20 Hz); 5.03-4.83(m, 4H); 4.44 (t, 1H, J=6.45 Hz); 4.1 (d, 1H, J=7.15 Hz); 3.70 (m, 1H);3.52 (m, 1H); 2.98 (s, 3H); 2.90-2.68 (m, 4H); 0.92 (d, 3H, J=6.9 Hz),0.89 (d, 3H, J=6.7 Hz).

LCMS (M-H⁺)=738.3

Example 28 Synthesis of compound 56(Z-Asp(β-tert-Butyl)-Indanylglycine-Val-Asp(β-tert-Butyl)methyl vinylsulfone)

The Z-Asp(β-tert-Butyl)-Indanylglycine-Val-OH (21 mg, 0.0353 mmol) isdissolved in a mix of dichloromethane and DMF (0.44 ml/0.15 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (10 μl) followed by isobutyl chloroformate (9 μl). Themixture was stirred at −15° C. for 10 minutes. Next,Asp(β-tert-butyl)methyl vinyl sulfone tosyl salt (15 mg, 1 eq) was addedin one shot, followed by N-methyl morpholine (10 μl). The mixture wasstirred 35 minutes at −15/−20° C. and then diluted with 5 ml ofdichloromethane and quenched with 1.5 ml of a saturated solution ofsodium bicarbonate. After that, the organic layer was washed with sodiumbicarbonate and dried over magnesium sulphate. The solvent wasevaporated to dryness. The obtained residue was purified on silica gel(ethyl acetate/hexane: 40% then dichloromethane/methanol: 5 to 15%) toafford 10 mg ofZ-Asp(β-tert-Butyl)-indanylglycine-Val-Asp(β-tert-butyl)-methyl vinylsulfone.

Example 30 Synthesis of compound 57 (Z-Asp-Indanylglycine-Val-Aspmethylvinyl sulfone)

Z-Asp(β-tert-Butyl)-Indanylglycine-Val-Asp(β-tert-butyl)-methyl vinylsulfone (11 mg) was dissolved in dichloromethane (0.26 ml), followed byaddition of trifluoroacetic acid (0.36 ml). The mixture was stirred atroom temperature overnight (15 h). It was then quenched with diethylether (5 ml) and the solvent was removed under vacuum. The obtainedresidue was diluted again with ether and the process was repeated twice.The precipitate was washed with 2*1 ml of ether, dried to give 9 mg ofthe desired compound.

NMR ¹H (DMSO, 400 MHz) δ: 12.35 (bs, 2H, 2*CO₂H); 8.21 (d, 1H, J=7.78Hz); 8.06 (m, 1H); 7.89 (d, 1H, J=7.34 Hz); 7.61 (d, 1H, J=7.10 Hz);7.34-7.31 (m, 5H); 7.17-6.95 (m, 4H); 6.75 (dd, 1H, J=15.33 Hz, J=4.8Hz); 6.64 (d, 1H, J=15.58 Hz); 4.99 (s, 2H); 4.81 (m, 1H); 4.46-4.40 (m,2H); 4.12 (t, 1H, J=6.28 Hz); 2.97 (s, 3H); 2.82-2.44 (m, 9H); 0.85 (t,6H, J=5.95 Hz).

Example 32 Synthesis of compound 58(Z-Asp(β-tert-Butyl)-Glu(β-tert-Butyl)-Val-Asp(β-tert-Butyl)methyl vinylsulfone)

The Z-Asp(β-tert-Butyl)-Glu(β-tert-Butyl)-Val-OH (18 mg, 0.029 mmol) isdissolved in a mix of dichloromethane and DMF (0.39 ml/0.13 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (8 μl) followed 3 min latter with isobutyl chloroformate (7μl). The mixture was stirred at −15° C. for 10 minutes. Next, Asp(β-tert-butyl)methyl vinyl sulfone tosyl salt (13 mg) was added in oneshot, followed by N-methyl morpholine (8 μl). The mixture was stirred 35minutes at −15/−20° C. and then diluted with 5 ml of dichloromethane andquenched with 1.5 ml of a saturated solution of sodium bicarbonate.After that, the organic layer was dried over magnesium sulphate. Thesolvent was evaporated to dryness. The obtained residue was purified onsilica gel (ethyl acetate/hexane: 40% then dichloromethane/methanol: 5to 15%) to afford 24 mg of the desired compound.

Example 33 Synthesis of compound 59 (Z-Asp-Glu-Val-Aspmethyl vinylsulfone)

Z-Asp(β-tert-Butyl)-Glu(β-tert-Butyl)-Val-Asp(β-tert-butyl)-methyl vinylsulfone (22.7 mg) was dissolved in dichloromethane (0.55 ml), followedby addition of trifluoroacetic acid (0.7 ml). The mixture was stirred atroom temperature overnight (15 h). It was then quenched with diethylether (7 ml) and the solvent was removed under vacuum. The obtainedresidue was diluted again with ether (5 ml) and the process was repeatedtwice. The precipitate was washed with 2*1 ml of ether, dried to give 19mg of the desired compound.

Example 34 Synthesis of compound 60 Z-Val-Asp(β-tert-Butyl)methyl vinylsulfone

The Z-Val-OH (15 mg, 0.059 mmol) is dissolved in a mix ofdichloromethane and DMF (0.5 ml/0.2 ml). The mixture was allowed toreach −15/−20° C. before adding N-methyl morpholine (10 μl) followed byisobutyl chloroformate (9 μl). The mixture was stirred at −15° C. for 10minutes. Next, Asp (β-tert-butyl)methyl vinyl sulfone tosyl salt (25 mg,1 eq) was added in one shot, followed by N-methyl morpholine (10 μl).The mixture was stirred 35 minutes at −15/−20° C. and then diluted with5 ml of dichloromethane and quenched with 1.5 ml of a saturated solutionof sodium bicarbonate. After that, the organic layer was dried overmagnesium sulphate. The solvent was evaporated to dryness. The obtainedresidue was purified on silica gel (gradient: methanol/dichloromethane:0 to 15%) to afford 24 mg of Z-Val-Asp-methyl vinyl sulfone.

NMR ¹H (CD₃OD, 400 MHz) δ: 7.38-7.29 (m, 5H); 6.82 (m, 1H); 6.69 (m,1H); 5.10 (m, 2H); 4.96 (m, 1H); 3.91 (m, 1H); 2.94 (s, 3H); 2.8-2.6 (m,2H); 2.1-2.0 (m, 1H); 1.43 (s, 9H); 0.95 (t, 6H, J=6.7 Hz).

Example 35 Synthesis of compound 61 (Z-Val-Asp-methyl vinyl sulfone)

Z-Val-Asp(β-tert-butyl)-methyl vinyl sulfone (25 mg) was dissolved indichloromethane (0.6 ml), followed by addition of trifluoroacetic acid(0.45 ml). The mixture was stirred at room temperature overnight (15 h).It was then quenched with diethyl ether (7 ml), the solvent was removedunder vacuum. The obtained residue was diluted again with ether and theprocess was repeated twice. The precipitate was washed with 2*1 ml ofether, dried to give 22 mg of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 7.38-7.27 (m, 5H); 6.86 (m, 1H); 6.68 (m,1H); 5.11 (m, 2H); 5.03 (m, 1H); 3.92 (m, 1H); 2.94 (s, 3H); 2.88-2.59(m, 2H); 2.09-2.01 (m, 1H); 0.95 (t, 6H, J=6.49 Hz).

Example 36 Synthesis of compound 62(Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)phenyl vinylsulfone)

The Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-OH (19 mg, 0.0306 mmol) isdissolved in a mix of dichloromethane and DMF (0.39 ml/0.13 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (8 μl) followed by isobutyl chloroformate (7 μl). The mixturewas stirred at −15° C. for 10 minutes. Next, Asp (β-tert-butyl)phenylvinyl sulfone tosyl salt (11 mg) was added in one shot, followed byN-methyl morpholine (8 μl). The mixture was stirred 35 minutes at−15/−20° C. and then diluted with 5 ml of dichloromethane and quenchedwith 1.5 ml of a saturated solution of sodium bicarbonate. After that,the organic layer was dried over magnesium sulphate. The solvent wasevaporated to dryness. The obtained residue was purified on silica gel(gradient: ethyl acetate/hexane: 5 to 100%) to afford 13.7 mg of thedesired compound.

Example 37 Synthesis of compound 63(Z-Asp-Ala(2′-quinolyl)-Val-Aspphenyl vinyl sulfone)

Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)-phenyl vinylsulfone (13.7 mg) was dissolved in dichloromethane (0.3 ml), followed byaddition of trifluoroacetic acid (0.4 ml). The mixture was stirred atroom temperature overnight (15 h). It was then quenched with diethylether (5 ml), the solvent was removed under vacuum. The obtained residuewas diluted again with ether and the process was repeated twice. Theprecipitate was washed with 2*1 ml of ether, dried to give 10 mg of thedesired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.58 (m, 1H); 8.11 (d, 1H, J=8.75 Hz); 8.04(d, 1H, J=7.99 Hz); 7.85 (d, 3H, J=7.25 Hz); 7.72-7.6 (m, 3H); 7.56 (t,2H, J=7.83 Hz); 7.31-7.28 (m, 5H); 6.93 (dd, 1H, J=15.24 Hz, J=5.15 Hz);6.65 (d, 1H, J=14.87 Hz); 4.97-4.80 (m, 4H); 4.40 (m, 1H); 4.21-4.02 (m,1H); 3.63-3.50 (m, 1H); 3.47-3.44 (m, 1H); 2.82-2.6 (m, 4H); 2.15-1.98(m, 1H); 0.81 (t, 6H, J=6.38 Hz).

LCMS (M-H+)=800.5

Example 38 Synthesis of compound 64(Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)phenoxy vinylsulfone)

The Z-Asp(β-tert-Butyl)-Al(2′-quinolyl)-Val-OH (10 mg, 0.016 mmol) isdissolved in a mix of dichloromethane and DMF (0.24 ml/0.080 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (5 μl) followed 3 min latter with isobutyl chloroformate (5μl). The mixture was stirred at −15° C. for 10 minutes. Next, Asp(β-tert-butyl)phenoxy vinyl sulfone tosyl salt (11.3 mg, 0.023 mmol) wasadded in one shot, the vial was washed with DMF (0.04 ml), followed bythe addition of N-methyl morpholine (5 μl). The mixture was stirred 35minutes at −15/−20° C. and then diluted with 5 ml of dichloromethane andquenched with 1.5 ml of a saturated solution of sodium bicarbonate.After that, the organic layer was dried over magnesium sulphate. Thesolvent was evaporated to dryness. The obtained residue was purified onsilica gel (gradient: ethyl acetate/hexane: 12 to 100%) to afford 4.7 mgof the desired compound.

Example 39 Synthesis if compound 65(Z-Asp-Ala(2′-quinolyl)-Val-Aspphenoxy vinyl sulfone)

Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)-phenoxy vinylsulfone (4.7 mg) was dissolved in dichloromethane (0.15 ml), followed byaddition of trifluoroacetic acid (0.2 ml). The mixture was stirred atroom temperature overnight (15 h). It was then quenched with diethylether (5 ml) and the solvent was removed under vacuum. The obtainedresidue was diluted again with ether (5 ml) and the process was repeatedtwice. The precipitate was washed with 2*1 ml of ether, dried to give 4mg of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.25 (d, 1H, J=7.9 Hz); 8.05 (d, 1H, J=8.66Hz); 7.88 (d, 1H, J=8.25 Hz); 7.63 (d, 1H, J=6.82 Hz); 7.53 (t, 1H,J=7.46 Hz); 7.45-7.33 (m, 3H); 7.33-7.20 (m, 8H); 6.75 (dd, 1H, J=15.33Hz, J=4.33 Hz); 6.65 (d, 1H, J=15.55 Hz); 5.1-4.8 (m, 4H); 4.49 (t, 1H,J=6.17 Hz); 4.06 (d, 1H, J=6.45 Hz); 3.6-3.4 (m, 2H); 2.9 (dd, 1H,J=17.02 Hz, J=5.39 Hz); 2.73 (dd, 1H, J=16.57 Hz, J=6.53 Hz); 2.59 (d,2H, J=6.97 Hz); 2.08 (m, 1H); 0.75 (m, 6H).

Example 40 Synthesis of compound 66(Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-Asp(β-tert-Butyl)morpholinevinyl sulfone)

Z-Asp(β-tert-Butyl)-Ala(2′-quinolyl)-Val-OH (13 mg, 0.0209 mmol) isdissolved in a mix of dichloromethane and DMF (0.28/0.094 ml). Themixture was allowed to reach −15/−20° C. before adding N-methylmorpholine (6 μl) followed by isobutyl chloroformate (5 μl). The mixturewas stirred at −15° C. for 10 minutes. Next, Asp (β-tert-butyl)morpholine vinyl sulfone tosyl salt (10.2 mg, 1 eq) was added in oneshot, followed by N-methyl morpholine (6 μl). The mixture was stirred 35minutes at −15/−20° C. and then diluted with 5 ml of dichloromethane andquenched with 1.5 ml of a saturated solution of sodium bicarbonate.After that, the organic layer was washed with sodium bicarbonate anddried over magnesium sulphate. The solvent was evaporated to dryness.The obtained residue was purified on silica gel (gradient: ethylacetate/hexane: 12 to 100%) to afford 10 mg of the desired compound.

Example 41 Synthesis of compound 67(Z-Asp-Ala(2′-quinolyl)-Val-Aspmorpholine vinyl sulfone)

Z-Asp(β-tert-Butyl)-Al(2′-quinolyl)-Val-Asp(β-tert-Butyl)-morpholinevinyl sulfone (4.7 mg) was dissolved in dichloromethane (0.15 ml),followed by addition of trifluoroacetic acid (0.2 ml). The mixture wasstirred at room temperature overnight (15 h). It was then quenched withdiethyl ether (5 ml) and the solvent was removed under vacuum. Theobtained residue was diluted again with ether and the process wasrepeated twice. The precipitate was washed with 2*1 ml of ether, driedto give 4 mg of the desired compound.

NMR ¹H (CD₃OD, 400 MHz) δ: 8.39 (s, 1H); 8.08 (d, 1H, J=8.56 Hz); 7.94(d, 1H, J=8.29 Hz); 7.74 (m, 1H); 7.66-7.55 (m, 2H); 7.31-7.29 (m, 5H);6.69 (dd, 1H, J=14.98 Hz, J=4.59 Hz); 6.41 (d, 1H, J=14.86 Hz); 5.0-4.7(m, 4H); 4.45 (m, 1H); 4.05 (d, 1H, J=6.65 Hz); 3.70-3.64 (m, 4H);3.55-3.44 (m, 2H); 3.16-3.08 (m, 4H); 2.89-2.70 (m, 4H); 2.17-2.07 (m,1H); 0.85-0.81 (m, 6H).

LCMS (M-H+)=809.6

Example 42 Synthesis of compound 68(Z-Asp-Indanylglycine-Val-Aspisopropyl vinyl sulfone)

This compound was synthesized from Asp(β-tert-butyl)isopropyl vinylsulfone tosyl salt (43) and Cbz-Asp(O-tBu)Indanylglycine-Val-OH (12) viaanhydride mixte method as for Z-Asp-Indanylglycine-Val-Aspmethyl vinylsulfone (57).

Example 43 Synthesis of compound 69 (Z-Asp-Phg-Val-Asp-phenylvinylsulfone)

This compound was synthesized from Z-Asp(β-tert-Butyl)-Phg-Val-OH (16)and Asp(OtBu)-Vinyl phenyl sulfone tosyl salt (37) via anhydride mixtemethod as for Z-Asp-Ala(2′-quinolyl)-Val-Aspphenyl vinyl sulfone (63).

Example 44 Synthesis of compound 70 (Z-Asp-(D, LAla(2′-quinolyl))-Val-Aspphenyl vinylsulfone)

This compound was synthesized as the correspondingZ-Asp-Ala(2′-quinolyl)-Val-Aspphenyl vinyl sulfone (63).

Example 45 Synthesis of compound 71 (Z-Asp-(D, LAla(2′-quinolyl))-Val-Aspmethyl vinylsulfone)

This compound was synthesized as the correspondingZ-Asp-Ala(2′-quinolyl)-Val-Aspmethyl vinyl sulfone (55).

Example 46 Synthesis of compound 76 (Z-Asp-Tyr-Val-Aspmethyl vinylsulfone)

This compound was synthesized from Z-Asp-Tyr(OtBu)-Val-OH (75) andAsp(OtBu)-Vinyl methy sulfone tosyl salt (33) via anhydride mixte methodas for Z-Asp-Phg-Val-Aspmethyl vinyl sulfone (53).

Z-Asp-Tyr(OtBu)-Val-OH was synthesized asCbz-Asp(O-tBu)-Glu(O-tBu)-Val-OH (20).

Example 47 Synthesis of compound 82 (Z-Tyr-Glu-Val-Aspmethyl vinylsulfone)

This compound was synthesized from Z-Tyr(OtBu)-Glu(OtBu)-Val-OH (80) andAsp(OtBu)-Vinyl methy sulfone tosyl salt (33) via anhydride mixte methodas for Z-Asp-Phg-Val-Aspmethyl vinyl sulfone (53).

Z-Tyr(OtBu)-Glu(OtBu)-Val-OH (80) was synthesized asCbz-Asp(O-tBu)-Glu(O-tBu)-Val-OH (20).

Example 48 Synthesis of compound 88 (Z-Asp-Trp-Val-Aspmethyl vinylsulfone)

This compound was synthesized from Z-Asp(OtBu)-Trp-Val-OH (86) andAsp(OtBu)-Vinyl methy sulfone tosyl salt (33) via anhydride mixte methodas for Z-Asp-Phg-Val-Aspmethyl vinyl sulfone (53).

Example 49 Synthesis of compound 85 (Z-Asp-Ala(2′-pyridyl)-Val-Aspmethylvinylsulfone)

This compound was synthesized from Z-Asp(OtBu)-Ala(2′-pyridyl)-Val-OH(83) and Asp(OtBu)-Vinyl phenyl sulfone tosyl salt (37) via anhydridemixte method as for Z-Asp-Ala(2′-quinolyl)-Val-Aspphenyl vinyl sulfone(63).

Example 50 Selectivity of exemplary compounds for caspase-3 relative tocaspase-1, caspase-5, caspase-7, and caspase-9

Selectivity of compound 55, compound 63, compound 48, compound 57,compound 88 toward caspase-1 (pro-inflammatory group), caspase-5 (groupI), caspase-9 (group II) and caspase-3 and caspase-7 (group III) wasevaluated by using fluorometric methods using the Caspase-1, -3, -5, -7,-9 Inhibitor Drug Screening Kit™ (Catalog #: K151-100, K153-100,K155-100, K157-100, K159-100 respectively, BioVision™). Briefly, usinginstructions of the manufacturer, a wide range of differentconcentrations of the compound: 3333, 1000, 333, 100, 33, 10, 3, and 1nM (final concentration) was added directly to the reaction mixturescontaining the substrate and the enzyme in a final volume of 10 μl.After a 30-minute incubation at 37° C., the liberation of AFC wasmeasured as an endpoint assay using the Flexstation3™ (MolecularDevices) with an excitation wavelength of 400 nm and an emissionwavelength of 505 nm. The level of inhibition of caspase-1, -3, -5, -7,-9 activity was determined by comparison of the relative fluorescenceintensity in samples with or without the compound. Results aresummarized in Table 2 herein after.

As shown in Table 2, Compound 55 showed an inhibitory effect on bothcaspase-3 and -7 activity. However, based on the IC50 values calculated,it was about 200 fold more selective of Caspase-3 over Caspase-7 (seeTable 2). No significant inhibitory activity of the compound wasobserved for caspase-1, -5, and -9 at the tested dose-range.

Inhibition of Caspase-1, -5, and -9 was achieved (25%, 21%, and 57%respectively) but at extremely high concentration (about 10,000 nM).

Similar to Compound 55, Compound 63 also showed high selectivity incaspase-3 activity inhibition relative to caspase-1, -5, -7, and -9.Compound 63 also showed an inhibitory effect on caspase-3 and -7, withabout a 50 fold selectivity of caspase-3 over caspase-7. No significantinhibitory activity was observed for caspase-1, -5, and -9 at the testeddose-range of Compound 63. The data indicates that both Compound 55 andCompound 63 are highly potent and selective compounds for caspase-3activity inhibition.

Compound 48 was able to inhibit specific groups of caspases. Thecompound was able to inhibit caspase-3, -7, and -9 with IC50 values ofabout 8-30 nM, 0.4-0.9 uM and 1-1.8 uM respectively. Thus Compound 48shows inhibition of Group III caspases (caspase-3 and -7) with highpotency, and inhibition of Group II caspase (caspase-9) with a lower yetsignificant efficacy.

From the inhibition profile of Compound 57 on caspase-1, -3, -5, -7, and-9 activity, it can be appreciated that Compound 57 is a potentinhibitor of Group III caspases (caspase-3 and -7). Compound 57inhibited caspase-3 and -7 activities with IC50 values of about 30-90 nMand about 180-300 nM respectively. Therefore, data indicates thatCompound 48 and Compound 57 are not specific inhibitors for caspase-3activity, but are in fact inhibitors targeted at specific groups ofcaspases.

Compound 88 has a dual inhibition effect on both caspase-1 and caspase-3activity. It is a potent inhibitor of caspase-3 activity, with an IC50value of about 30-90 nM. It also inhibits caspase-1 activity with anIC50 value of about 0.6-1.2 uM.

Example 51 In Vitro Inhibitory Activity of Caspase-3 Inhibitors

To test the efficacy of caspase-3 inhibitors at the cellular level, theability of selected compounds to inhibit the proteolytic cleavage ofPARP (poly ADP-ribose polymerase) was evaluated in live Hela cells.

Briefly, in this assay Hela cells are seeded in 96 well plates andincubated for 4 hours with staurosporine, a well characterized inducerof apoptosis, alone or together with different concentrations ofcompound (50, 25, 10 and 3 uM). After formaldehyde-based fixation, thecells are stained with a fluorescein-labeled anti-cleaved PARP antibody(Cell signaling, Cat#: 9547) and counterstained with Hoechst33342(Invitrogen, Cat#: H3570) to mark all nuclei. Fluorescence images aretaken on a Cellomics™ microscope system (Thermo Scientific, Pittsburgh,USA) with the Hoechst stain in the blue channel and the cleaved PARPantibody stain in the green channel. The percentage of cleaved PARPpositive cells is determined by calculating the ratio between nucleiwith a cleaved PARP antibody staining above a certain threshold and all(Hoechst positive) nuclei. The efficacy of caspase-3 inhibition isdetermined by calculating the ratio between cleaved PARP positive cellsafter staurosporine incubation together with compounds and staurosporineincubation without compounds. Results are summarized in Table 2.

As shown in Table 2, results of this assay show that compounds thatinhibited Caspase-3 activity in the enzymatic assay with an IC50 below100 nM were generally also effective in inhibiting PARP cleavage invitro, although a major factor contributing to a compound'sactivity/effectivity in this assay is the compound's cell membranepermeability coefficient (the higher the permeability coefficient, thegreater the amount of the compound in the media reaching inside thecell). Certain modifications in the molecular composition of thecompounds improved the inhibitory activity further; for example Compound51, Compound 53, Compound 57, and Compound 76 reduced the percentage ofPARP positive cells after staurosporine treatment by more than 50%, withCompound 51 and Compound 57 being the most effective compounds reachingvalues of about 65% inhibition, and Compound 53 also being highlyeffective relative to DEVD-fmk (positive control compound with lowcaspase-3 selectivity but highly cell membrane permeable with generalcaspase inhibitory activity), and Compounds 48, 55, and 59 also showingeffectivity especially relative to DEVD-fmk. These results indicate thatinhibitory compounds identified in a primary enzymatic assay screenretain their activity in a cellular environment and that molecularmodifications allow to further improve their activity as caspase-3inhibitors.

TABLE 2 Activity assays for caspase inhibitors. Values given areapproximations of the average values obtained from the assays. % inhibi-tion of % inhibi- PARP % inhibi- tion of cleavage tion of PARP at 50 uMinhibi- cleavage DEVD- tion with % of Caspase enzymatic activity, IC50(uM) at 50 uM FMK compound IC50 Compound Caspase Caspase Caspase CaspaseCaspase com- (positive relative to (uM) No Name 1 3 5 7 9 pound control)DEVDFMK Glo3/7 48 Z-Asp-Ala(2′-quinolyl)-Val-Asp alpha >3.33 0.01 >3.330.4 1.2 45 70 65 0.1 chlorovinyl methylsulfone 50Tosyl-Ala(2′-quinolyl)-Val-Asp alpha >3.33 >3.33 chlorovinylmethylsulfone 36 Boc-Asp(O-tBu)VSphenyl >2 37 Asp(O-tBu)VSphenyl salt >216 Z-Asp(O-tBu)-Phg-Val-OH >2 51Z-Asp(O-Methyl)-Indanylglycine-Val- >3.0 1 >3.0 >3.0 >3.0 65 80 80Asp(O-Methyl)VSmethyl 53 Z-Asp-Phg-Val-AspVSmethyl 2 0.06 >10 0.8 >10 5570 80 0.1 93 AspVSmethyl salt >10 33 Asp(O-tBu)VSmethyl salt >10 71Z-Asp-d,l Ala(2′-quinolyl)-Val- >10 0.1 >10 8.9 >10 AspVSmethyl 55Z-Asp-Ala(2′-quinolyl)-Val-AspVSmethyl >10 0.01 >10 1.4 >3.33 40 65 600.1 65 Z-Asp-Ala(2′-quinolyl)-Val-AspVSphenoxy 1.2 0.01 >3.33 0.4 0.3 3075 40 0.2 70 Z-Asp-d,l Ala(2′-quinolyl)-Val-AspVSphenyl 0.3 >10 >10 >1063 Z-Asp-Ala(2′-quinolyl)-Val-AspVSphenyl >3.33 0.04 >3.33 1.8 >3.33 1070 15 0.9 66 Z-Asp-Ala(2′-quinolyl)-Val-AspVSmorpholine 0.2 25 85Z-Asp-Ala(2′-pyridyl)-Val-AspVSphenyl >3.33 0.03 >3.33 1.0 >3.33 40 69Z-Asp-Phg-Val-AspVSphenyl 0.1 76 Z-Asp-Tyr-Val-AspVSmethyl >3.330.03 >3.33 0.7 >3.33 55 90 60 0.04 57Z-Asp-Indanylglycine-Val-AspVSmethyl 0.9 0.03 >3.33 0.2 >3.33 65 90 700.1 88 Z-Asp-Trp-Val-AspVSmethyl 0.7 0.04 >3.33 1.1 >3.33 45 90 50 59Z-Asp-Glu-Val-AspVSmethyl 1.4 0.02 >3.33 0.04 0.5 45 65 70 61Z-Val-AspVSmethyl >3.33 >3.33 >3.33 >3.33 >3.33 5 70 5 68Z-Asp-Indanylglycine-Val-AspVSisopropyl >3.33 0.2 >3.33 1.5 >3.33 30 6050 96 Z-Tyr-Val-Ala-AspVSphenyl 1.3 >10 >3.33 >10 >10 82Z-Tyr-Glu-Val-AspVSmethyl 0.5 0.3 >3 >5 1.6 DEVD-fmk (positive controlcompound) 0.4 0.6 >3.33 1 0.5 85 (20 uM IC50)

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound of Formula I:

wherein a is 0 or 1; b is 0 or 1 provided that when b is 0, a is 0; Ais 1) H, 2) C₁-C₆ alkyl, 3) aryl, 4) heteroaryl, 5) heterocyclyl, 6)R³—C(O)—, 7) R³—OC(O)—; 8) R³—C(O)O—, or 9) R³—S(O)₂—; P2, P3, P4 and,when present, P5 and PX, are any (D) or (L) amino acid residue; the line“-” when located between P2, P3, P4, P5 or PX represents a peptide bondor a peptidomimetic bond; the wavy line represents either cis or transorientation of R¹ and R²; R¹ is 1) aryl, 2) heteroaryl, 3) heterocyclyl,4) C₂-C₆ alkene-R²⁰, 5) SO₂R⁵, 6) SO₃R⁵, 7) SOR⁵, 8) SONHR⁵, 9) SO₂NHR⁵,10) CN, 11) CO₂R⁵, 12) COR⁵, 13) PO₃R⁵, 14) PO(OR⁵)₂, or 15) PO(OR⁵),wherein the aryl, the heteroaryl, or the heterocyclyl are optionallysubstituted with one or more R³⁰; R² is 1) R¹; or 2) H, 3) halogen, 4)haloalkyl, 5) C₁-C₆ alkyl, 6) C₂-C₆ alkene, 7) C₃-C₇ cycloalkyl, 8) OR⁹;9) OCOR⁶, 10) OCO₂R⁶, 11) NR⁷R⁸, 12) NHSO₂R⁶, 13) NHCOR⁶, 14) aryl, 15)heteroaryl, or 16) heterocyclyl; R³ is 1) C₁-C₆ alkyl, 2) aryl, 3)heteroaryl, or 4) heterocyclyl; R⁴ is 1) H, or 2) C₁-C₆ alkyl; R⁵ is 1)H, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkene, 4) C₃-C₇ cycloalkyl, 5) aryl, 6)heteroaryl, 7) heterocyclyl, or 8) any optionally protected (D) or (L)amino acid residue, or non-natural amino acid residue; R⁶ is 1) any (D)or (L) amino acid residue or non-natural amino acid residue, 2) C₁-C₆alkyl, 3) C₃-C₇ cycloalkyl, 4) aryl, 5) heteroaryl, or 6) heterocyclyl,in which the alkyl or the cycloalkyl are optionally substituted with oneor more R¹⁰ substituents; and in which the aryl, heteroaryl orheterocyclyl are optionally substituted with one or more R²⁰substituents; R⁷ and R⁸ are independently selected from: 1) H, 2) C₁-C₆alkyl, 3) C₃-C₇ cycloalkyl, 4) haloalkyl, 5) aryl, 6) heteroaryl, or 7)heterocyclyl, wherein the alkyl and the cycloalkyl are optionallysubstituted with one or more R¹⁰ substituents, and the aryl, theheteroaryl and the heterocyclyl are optionally substituted with one ormore R²⁰ substituents; R⁹ is 1) H, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl,4) aryl, 5) heteroaryl, or 6) heterocyclyl, in which the alkyl or thecycloalkyl are optionally substituted with one or more R¹⁰ substituents;and in which the aryl, heteroaryl or heterocyclyl are optionallysubstituted with one or more R²⁰ substituents; R¹⁰ is independentlyselected from: 1) halogen, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4)haloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, 8) OR⁹, 9)S(O)_(m)R⁹, 10) NR⁷R⁸, 11) COR⁹, 12) C(O)OR⁹, 13) OC(O)R⁹, 14) SC(O)R⁹,15) CONR⁷R⁸, or 16) S(O)₂NR⁷R⁸; R²⁰ is independently selected from: 1)halogen, 2) NO₂, 3) CN, 4) C₁-C₆ alkyl, 5) haloalkyl, 6) C₃-C₇cycloalkyl, 7) OR⁷, 8) NR⁷R⁸, 9) SR⁷, 10) aryl, 11) heteroaryl, 12)heterocyclyl, 13) SO₂R⁵, 14) SO₃R⁵, 15) SOR⁵, 16) SONHR⁵, 17)SO₂NHR⁵,18) PO₃R⁵, 19) PO(OR⁵)₂, 20) PO(OR⁵), 21) COR⁷, 22) CO₂R⁷, 23)S(O)_(m)R⁷, 24) CONR⁷R⁸, or 25) S(O)₂NR⁷R⁸, wherein the alkyl and thecycloalkyl are optionally substituted with one or more R⁶ substituents;and wherein the aryl, the heteroaryl, or the heterocyclyl are optionallysubstituted with one or more R³⁰; R³⁰ is 1) NO₂, 2) C₂-C₆ alkene-R²⁰, 3)SO₂R⁵, 4) SOR⁵, 5) SONHR⁵, 6) SO₂NHR⁵, 7) CN, 8) CO₂R⁵, 9) COR⁵, 10)PO₃R⁵, 11) PO(OR⁵)₂, or 12) PO(OR⁵); or a prodrug, or a pharmaceuticallyacceptable salt, or the compound is labeled with a detectable label oran affinity tag thereof.
 2. The compound, according to claim 1, in whichA is H.
 3. The compound, according to claim 1, in which A is PhCH₂OC(O)—4. The compound according to claim 1, in which R⁴ is H, CH₃ or CH₃CH₂.5. The compound, according to claim 1, in which R⁴ is H.
 6. Thecompound, according to claim 1, in which a and b are both
 0. 7. Thecompound, according to claim 1, in which a is 0 and b is
 1. 8. Thecompound, according to claim 1, in which R¹ is in the transconfiguration.
 9. The compound, according to claim 1, R¹ is 1) SO₂R⁵, 2)SO₃R⁵; or 3) SOR⁵, wherein R⁵ is C₁-C₆ alkyl, aryl, or heterocyclyl. 10.The compound, according to claim 1, in which R² is 1) H, 2) halogen, 3)haloalkyl, 4) C₁-C₆ alkyl, or 5) C₃-C₇ cycloalkyl.
 11. The compound,according to claim 10, in which R² is H or Cl
 12. (canceled)
 13. Acompound of Formula IA

wherein a is 0 or 1; b is 0 or 1 provided that when b is 0, a is 0; Ais 1) H, 2) C₁-C₆ alkyl, 3) aryl, 4) heteroaryl, 5) heterocyclyl, 6)R³—OC(O)—; 7) R³—C(O)O—, or 8) R³—S(O)₂—; AA₂ is the (R) or (S) aminoacid side chain of Val, Leu, Pro, Met, Ala, Thr, His, Ser, Lys, or Ile;AA₃ is the (R) or (S) amino acid side chain of Trp, Tyr, Ala, Asp, Gln,Glu, Phe, Ser, Thr, Val, Tyr, Gly, Leu, His, or Ile; or AA₃ isphenylglycine, indanylglycine, or Ala-(2′-quinolyl); AA₄ is the (R) or(S) amino acid side chain of Asp, Ile, Leu, Glu, Ala, Val, Tyr, Trp,Phe, or Pro; AA₅, when present, is the (R) or (S) amino acid side chainof Val or Leu; AA_(X), when present, is the (R) or (S) amino acid sidechain of any D or L amino acid residue or the amino acid side chain ofthe non-natural amino acid residue; the wavy line represents either cisor trans orientation of R¹ and R²; R¹ is 1) aryl, 2) heteroaryl, 3)heterocyclyl, 4) C₂-C₆ alkene-R²⁰, 5) SO₂R⁵, 6) SO₃R⁵, 7) SOR⁵, 8)SONHR⁵, 9) SO₂NHR⁵, 10) CN, 11) CO₂R⁵, 12) COR⁵, 13) PO₃R⁵, 14)PO(OR⁵)₂, or 15) PO(OR⁵), wherein the aryl, the heteroaryl, or theheterocyclyl are optionally substituted with one or more R³⁰; R² is 1)R¹; or 2) H, 3) halogen, 4) haloalkyl, 5) C₁-C₆ alkyl, 6) C₂-C₆ alkene,7) C₃-C₇ cycloalkyl, 8) OR⁹; 9) OCOR⁶, 10) OCO₂R⁶, 11) NR⁷R⁸, 12)NHSO₂R⁶, 13) NHCOR⁶, 14) aryl, 15) heteroaryl, or 16) heterocyclyl; R³is 1) C₁-C₆ alkyl, 2) aryl, 3) heteroaryl, or 4) heterocyclyl; R⁴ is 1)H, or 2) C₁-C₆ alkyl; R⁵ is 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkene, 4)C₃-C₇ cycloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, or 8) anyoptionally protected (D) or (L) amino acid residue; R⁶ is 1) any (D) or(L) amino acid residue, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4) aryl, 5)heteroaryl, or 6) heterocyclyl, in which the alkyl or the cycloalkyl areoptionally substituted with one or more R¹⁰ substituents; and in whichthe aryl, heteroaryl or heterocyclyl are optionally substituted with oneor more R²⁰ substituents; R⁷ and R⁸ are independently selected from: 1)H, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4) haloalkyl, 5) aryl, 6)heteroaryl, or 7) heterocyclyl, wherein the alkyl and the cycloalkyl areoptionally substituted with one or more R¹⁰ substituents, and the aryl,the heteroaryl and the heterocyclyl are optionally substituted with oneor more R²⁰ substituents; R⁹ is 1) H, 2) C₁-C₆ alkyl, 3) C₃-C₇cycloalkyl, 4) aryl, 5) heteroaryl, or 6) heterocyclyl, in which thealkyl or the cycloalkyl are optionally substituted with one or more R¹⁰substituents; and in which the aryl, heteroaryl or heterocyclyl areoptionally substituted with one or more R²⁰ substituents; R¹⁰ isindependently selected from: 1) halogen, 2) C₁-C₆ alkyl, 3) C₃-C₇cycloalkyl, 4) haloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, 8)OR⁹, 9) S(O)_(m)R⁹, 10) NR⁷R⁸, 11) COR⁵, 12) C(O)OR⁹, 13) OC(O)R⁹, 14)SC(O)R⁹, 15) CONR⁷R⁸, or 16) S(O)₂NR⁷R⁸; R²⁰ is independently selectedfrom: 1) halogen, 2) NO₂, 3) CN, 4) C₁-C₆ alkyl, 5) haloalkyl, 6) C₃-C₇cycloalkyl, 7) OR⁷, 8) NR⁷R⁸, 9) SR⁷, 10) aryl, 11) heteroaryl, 12)heterocyclyl, 13) SO₂R⁵, 14) SO₃R⁵, 15) SOR⁵, 16) SONHR⁵, 17) SO₂NHR⁵,18) PO₃R⁵, 19) PO(OR⁵)₂, 20) PO(OR⁵), 21) COR⁷, 22) CO₂R⁷, 23)S(O)_(m)R⁷, 24) CONR⁷R⁸, or 25) S(O)₂NR⁷R⁸, wherein the alkyl and thecycloalkyl are optionally substituted with one or more R⁶ substituents;and wherein the aryl, the heteroaryl, or the heterocyclyl are optionallysubstituted with one or more R³⁰; R³⁰ is 1)NO₂, 2) C₂-C₆ alkene-R²⁰, 3)SO₂R⁵, 4) SOR⁵, 5) SONHR⁵, 6) SO₂NHR⁵, 7) CN, 8) CO₂R⁵, 9) COR⁵, 10)PO₃R⁵, 11) PO(OR⁵)₂, or 12) PO(OR⁵); or a prodrug, or a pharmaceuticallyacceptable salt, or the compound is labeled with a detectable label oran affinity tag thereof.
 14. The compound, according to claim 13,includes compounds of Formula IIA

wherein AA₂ is the amino acid side chain of Val, Leu, Pro, Met, Ala,Thr, His, Ser, Lys, or Ile; AA₃ is the amino acid side chain of Trp,Tyr, Ala, Asp, Gln, Phe, Ser, Thr, Val, Tyr, Gly, Leu; or AA₃ isphenylglycine, indanylglycine, or Ala-(2′-quinolyl); AA₄ is the aminoacid side chain of Asp or Trp; or wherein AA₂ is the amino acid sidechain of Thr, His, Val, Trp, Ile, or Ala AA₃ is the amino acid sidechain of Glu or AA₃ is Ala-(2′-quinolyl); AA₄ is the amino acid sidechain of Ile, Leu, Glu, Asp, Ala, Pro or Val; or wherein AA, is theamino acid side chain of Val, Ala, Thr, or His; AA₃ is the amino acidside chain of Glu, Gln, Asp, Ala, Gly, Thr, Val, Trp; or AA₃ isphenylglycine or indanylglycine; AA₄ is the amino acid side chain ofTyr, Trp, Phe, or Asp; and wherein A, R¹, R² and R⁴ are as defined inclaim
 13. 15. (canceled)
 16. The compound, according to claim 13,includes compounds of Formula IIIA

wherein AA₂ is the amino acid side chain of Ala, Ser, Lys or Val; AA₃ isthe amino acid side chain of Val, Glu, Thr, or Gln; AA₄ is the aminoacid side chain of Asp, or Leu; AA₅ is the amino acid side chain of Valor Leu; and wherein A, R¹, R² and R⁴ are as defined in claim
 13. 17.(canceled)
 18. The compound according to claim 1, wherein said compoundis selected from the group consisting of:


19. (canceled)
 20. A pharmaceutical composition for preventing and/ortreating a caspase-related disease in a subject in need thereof,comprising an effective amount of a compound according to claim
 1. 21. Amethod for preventing and/or treating a caspase-related disease in asubject in need thereof, comprising administering to said subject aneffective amount of a compound according to claim
 1. 22-23. (canceled)24. The method, according to claim 21 in which the caspase-relateddisease is selected from the group consisting of apoptosis mediateddiseases, IL-1 mediated diseases, inflammatory diseases, autoimmunediseases, autoinflammatory diseases, proliferative diseases, infectiousdiseases, degenerative diseases, retinal disorders, inflammatoryperitonitis, osteoarthritis, pancreatitis, asthma, respiratory distresssyndrome, rheumatoid arthritis, systemic lupus erythematous,scleroderma, Grave's disease, auto immune gastritis, diabetes,autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia,hepatitis, inflammatory bowel disease, crohn's disease, psoriasis,dermatitis, Graft vs host disease, organ transplant rejection,osteoporosis, leukemias and related disorders, multiple myeloma-relateddiseases, metastatic melanomas, Kaposi's sarcoma, sepsis, septic shock,Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebralischemia, epilepsy, myocardial ischemia, acute and chronic heartdisease, myocardial infarction, congestive heart failure,atherosclerosis, spinal muscular atrophy, amyotrophic lateral sclerosis,multiple sclerosis, HIV-related encephalitis, aging, neurological damagedue to stroke, ulcerative colitis, traumatic brain injury, spinal cordinjury, hepatitis-B, hepatitis-C, hepatitis-G, liver-related diseases,renal disease, and HIV infection.
 25. (canceled)
 26. A method oftreating excessive apoptosis affected by caspase activity in a cell or atissue, the method comprising: contacting the cell or tissue with aneffective amount of one or more compounds according to claim 1, so as totreat the excessive apoptosis. 27-29. (canceled)
 30. The compound,according to claim 1, in which the pharmaceutically acceptable salt isone that allows the compound to penetrate cells.